EP4114958A1 - Compositions et méthodes de traitement d'une hypoacousie non associée à l'âge chez un sujet humain - Google Patents

Compositions et méthodes de traitement d'une hypoacousie non associée à l'âge chez un sujet humain

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EP4114958A1
EP4114958A1 EP21715694.2A EP21715694A EP4114958A1 EP 4114958 A1 EP4114958 A1 EP 4114958A1 EP 21715694 A EP21715694 A EP 21715694A EP 4114958 A1 EP4114958 A1 EP 4114958A1
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amino acids
acid substitutions
amino acid
amino
acids
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Emmanuel John Simons
Robert NG
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Akouos Inc
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Akouos Inc
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Definitions

  • the present disclosure relates generally to the use of nucleic acids to treat hearing loss in a human subject.
  • the ear is a complex organ, classically described as including the outer ear, the middle ear, the inner ear, the hearing (acoustic) nerve and the auditory system (which processes sound as it travels from the ear to the brain). In addition to detecting sound, the ear also helps to maintain balance. Thus, disorders of the inner ear can cause hearing loss, tinnitus, vertigo and imbalance. Vertigo is a hallucination of motion, and is the cardinal symptom of vestibular system disease. Vertigo can be caused by problems in the inner ear or central nervous system.
  • vertigo common inner ear causes of vertigo include: vestibular neuritis (sudden, unilateral vestibular loss), Meniere's disease (episodic vertigo), benign paroxysmal positional vertigo (BPPV), and bilateral vestibular loss.
  • Common central nervous system causes of vertigo include: post-concussion syndrome, cervical vertigo, vestibular migraine, cerebrovascular disease, and acoustic neuroma.
  • Hearing loss is one of the most common human sensory deficits, and can occur for many reasons. Some people may be born with hearing loss while others may lose their hearing slowly over time. Presbycusis (also spelled presbyacusis) is age-related hearing loss. Approximately 36 million American adults report some degree of hearing loss, and one in three people older than 60 and half of those older than 85 experience hearing loss.
  • Hearing loss can be the result of environmental factors or a combination of genetic and environmental factors. About half of all people who have tinnitus— phantom noises in their auditory system (ringing, buzzing, chirping, humming, or beating)— also have an over-sensitivity to/reduced tolerance for certain sound frequency and volume ranges, known as hyperacusis (also spelled hyperacousis).
  • Williams syndrome also known as Williams-Beuren Syndrome
  • LIM kinase 1 Individuals with Williams Syndrome manifest hyperacusis and progressive hearing loss, and hyperacusis early onset suggests that it could be associated with one of the deleted genes.
  • autoimmune inner ear disease is characterized by rapidly progressive bilateral sensorineural hearing loss, occurring when the body's immune system attacks cells in the inner ear that are mistaken for a virus or bacteria.
  • Nonsyndromic deafness is hearing loss that is not associated with other signs and symptoms.
  • syndromic deafness involves hearing loss that occurs with abnormalities in other parts of the body.
  • Most cases of genetic deafness 70 percent to 80 percent are nonsyndromic; the remaining cases are caused by specific genetic syndromes.
  • Hearing loss can be conductive (arising from the ear canal or middle ear), sensorineural (arising from the inner ear or auditory nerve), or mixed. Most forms of nonsyndromic deafness are associated with permanent hearing loss caused by damage to structures in the inner ear (sensorineural deafness). The great majority of human sensorineural hearing loss is caused by abnormalities in the hair cells of the organ of Corti in the cochlea. There are also very unusual sensorineural hearing impairments that involve the eighth cranial nerve (the vestibulocochlear nerve) or the auditory portions of the brain. In the rarest of these sorts of hearing loss, only the auditory centers of the brain are affected.
  • cortical deafness may occur, where sounds may be heard at normal thresholds, but the quality of the sound perceived is so poor that speech cannot be understood.
  • most sensorineural hearing loss is due to poor hair cell function.
  • the hair cells may be abnormal at birth, or damaged during the lifetime of an individual. There are both external causes of damage, like noise trauma and infection, and intrinsic abnormalities, like congenital mutations to genes that play an important role in cochlear anatomy or physiology.
  • Hearing loss that results from changes in the middle ear is called conductive hearing loss.
  • Some forms of nonsyndromic deafness involve changes in both the inner ear and the middle ear, called mixed hearing loss.
  • Hearing loss that is present before a child learns to speak is classified as prelingual or congenital.
  • Hearing loss that occurs after the development of speech is classified as postlingual.
  • Most autosomal recessive loci cause prelingual severe-to-profound hearing loss.
  • Nonsyndromic deafness can have different patterns of inheritance, and can occur at any age. Types of nonsyndromic deafness are named according to their inheritance patterns. Autosomal dominant forms are designated DFNA, autosomal recessive forms are DFNB, and X-linked forms are DFN. Each type is also numbered in the order in which it was described. For example, DFNA1 was the first described autosomal dominant type of nonsyndromic deafness.
  • each parent of an individual with autosomal recessive deafness is a carrier of one copy of the mutated gene, but is not affected by this form of hearing loss.
  • Another 20 percent to 25 percent of nonsyndromic deafness cases are autosomal dominant, which means one copy of the altered gene in each cell is sufficient to result in hearing loss. People with autosomal dominant deafness most often inherit an altered copy of the gene from a parent who has hearing loss.
  • X-linked pattern of inheritance which means the mutated gene responsible for the condition is located on the X chromosome (one of the two sex chromosomes).
  • Males with X-linked nonsyndromic deafness tend to develop more severe hearing loss earlier in life than females who inherit a copy of the same gene mutation.
  • a characteristic of X-linked inheritance is that fathers cannot pass X-linked traits to their sons.
  • Mitochondrial nonsyndromic deafness which results from changes to mitochondrial DNA, occurs in less than one percent of cases in the United States.
  • the altered mitochondrial DNA is passed from a mother to all of her sons and daughters.
  • ANSD Auditory neuropathy spectrum disorder
  • ANSD Auditory neuropathy spectrum disorder
  • the OTOF gene is the first gene identified for autosomal recessive non-syndromic ANSD, and mutations in OTOF have been found to account for approximately 5% of all cases of autosomal recessive nonsydromic hearing loss in some populations (Rodriguez -Ballesteros et al. 2008 Human Mut 29(6):823-831).
  • nonsyndromic deafness The causes of nonsyndromic deafness are complex.
  • researchers have identified more than 30 genes that, when altered, are associated with nonsyndromic deafness; however, some of these genes have not been fully characterized. Different mutations in the same gene can be associated with different types of hearing loss, and some genes are associated with both syndromic and nonsyndromic deafness.
  • genes associated with nonsyndromic deafness include, but are not limited to, ATP2B2, ACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB3, KCNQ4, LHFPL5, MYOIA, MY015A, MY06, MY07A, OTOF, PCDH15, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIOBP, USH1C, and WFS1.
  • Nonsyndromic Hearing Loss and Deafness DFNB1 (also called GJB2-related DFNB1 Nonsyndromic Hearing Loss and Deafness; Autosomal Recessive Deafness 1; Neurosensory Nonsyndromic Recessive Deafness 1).
  • Nonsyndromic hearing loss and deafness DFNB1 is characterized by congenital, non-progressive, mild-to-profound sensorineural hearing impairment. It is caused by mutations in GJB2 (which encodes the protein connexin 26) and GJB6 (which encodes connexin 30).
  • Diagnosis of DFNBl depends on molecular genetic testing to identify deafness-causing mutations in GJB2 and upstream cis-regulatory elements that alter the gap junction beta-2 protein (connexin 26). Molecular genetic testing of GJB2 detects more than 99% of deafness-causing mutations in these genes. Unlike some other forms of hearing loss, DFNBl nonsyndromic hearing loss and deafness does not affect balance or movement. The degree of hearing loss is difficult to predict based on which genetic mutation one has. Even if members of the same family are affected by DFNBl nonsyndromic hearing loss and deafness, the degree of hearing loss may vary among them.
  • Cx26 and Cx30 are the two major Cx isoforms found in the cochlea, and they coassemble to form hybrid (heteromeric and heterotypic) gap junctions (GJs) (Ahmad, et ak, Proc. Natl. Acad. Sci., 2007, 104(4): 1337-1341).
  • Nonsyndromic hearing loss and deafness is caused by a dominant-negative pathogenic variant in the GJB2 or GJB6 gene, altering either the protein connexin 26 (Cx26) or connexin 30 (Cx30), respectively, and is characterized by pre- or postlingual, mild to profound, progressive high-frequency sensorineural hearing impairment.
  • OTOF-related deafness is characterized by two phenotypes: prelingual nonsyndromic hearing loss and, less frequently, temperature- sensitive nonsyndromic auditory neuropathy (TS-NSAN).
  • TS-NSAN temperature- sensitive nonsyndromic auditory neuropathy
  • Another form of progressive hearing impairment is associated with a mutation in the otoferlin gene (e.g., a I1573T mutation or a P1987R mutation, and/or a E1700Q mutation), or is not temperature sensitive.
  • Pendred syndrome/DFNB4 (deafness with goiter) is an autosomal recessive inherited disorder, and accounts for 7.5% of all cases of congenital deafness. Pendred syndrome has been linked to mutations in the PDS gene (also known as DFNB4, EVA, PDS, TDH2B and solute carrier family 26, member 4, SLC26A4) on the long arm of chromosome 7 (7q31), which encodes the pendrin protein. Mutations in this gene also cause enlarged vestibular aqueduct syndrome (EVA or EVAS), another congenital cause of deafness; specific mutations are more likely to cause EVAS, while others are more linked with Pendred syndrome. (Azaiez, et al. (December 2007), Hum. Genet. 122 (5): 451-7).
  • Transmembrane protease, serine 3 is an enzyme encoded by the TMPRSS3 gene (also known as DFNB10, DFNB8, ECHOS1, and TADG12). The gene was identified by its association with both congenital and childhood onset autosomal recessive deafness. Mutations in TMPRSS3 are associated with postlingual and rapidly progressive hearing impairment.
  • the protein encoded by the TMPRSS3 gene contains a serine protease domain, a transmembrane domain, an LDL receptor-like domain, and a scavenger receptor cysteine-rich domain. Serine proteases are known to be involved in a variety of biological processes, whose malfunction often leads to human diseases and disorders.
  • This gene is expressed in fetal cochlea and many other tissues, and is thought to be involved in the development and maintenance of the inner ear or the contents of the perilymph and endolymph. This gene was also identified as a tumor associated gene that is overexpressed in ovarian tumors. Four alternatively spliced variants have been described, two of which encode identical products.
  • DFN3 deafness is caused by mutations in the POU3F4 gene, which is located on the X chromosome. In people with this condition, one of the small bones in the middle ear (the stapes) cannot move normally, which interferes with hearing. This characteristic sign of DFN3 is called stapes fixation. At least four other regions of the X chromosome are involved in hearing loss, but the responsible genes have not been discovered.
  • DFNB59 deafness, autosomal recessive 59
  • Pejvakin or PJVK is a 352 amino acid protein belonging to the gasdermin family in vertebrates.
  • DFNB59 is encoded by a gene that maps to human chromosome 2q31.2, essential for the proper function of auditory pathway neurons and outer hair cell function. Mutations in DFNB59 are believed to cause non-syndromic sensorineural deafness autosomal recessive type 59, a form of sensorineural hearing impairment characterized by absent or severely abnormal auditory brainstem response but normal otoacoustic emissions (auditory neuropathy or auditory dys-synchrony). DFNB59 shares significant similarity with DFNA5, indicating that these genes share a common origin.
  • Alport syndrome is caused by mutations in the COL4A3, COL4A4, and COL4A5 genes involved in collagen biosynthesis. Mutations in any of these genes prevent the proper production or assembly of the type IV collagen network, which is an important structural component of basement membranes in the kidney, inner ear, and eye.
  • One of the criteria used in diagnosis of Alport syndrome is bilateral sensorineural hearing loss in the 2000 to 8000 Hz range. The hearing loss develops gradually, is not present in early infancy and commonly presents before the age of 30 years.
  • DFNA2 nonsyndromic hearing loss is inherited as an autosomal dominant mutation in the KCNQ4 gene, which encodes the potassium voltage-gated channel subfamily KQT member 4 also known as voltage-gated potassium channel subunit Kv7.4.
  • DFNA2 nonsyndromic hearing loss is characterized by symmetric, predominantly high-frequency sensorineural hearing loss (SNHL) that is progressive across all frequencies. At younger ages, hearing loss tends to be mild in the low frequencies and moderate in the high frequencies; in older persons, the hearing loss is moderate in the low frequencies and severe to profound in the high frequencies.
  • JLNS Jervell and Lange-Nielsen syndrome
  • This condition is an autosomal recessive disorder that affects an estimated 1.6 to 6 in 1 million children, and is responsible for less than 10 percent of all cases of long QT syndrome. It has a markedly higher incidence in Norway and Sweden, up to 1 :200,000.
  • the proteins produced by the KCNE1 and KCNQ1 genes work together to form a potassium channel that transports positively charged potassium ions out of cells. The movement of potassium ions through these channels is critical for maintaining the normal functions of the inner ear and cardiac muscle.
  • EAST/SeSAME syndrome characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt waste, is believed to be caused by mutations in KCNJ10 inwardly rectifying potassium channels.
  • Bartter's syndrome with sensorineural deafness type 4 also known as Bartter syndrome IV or BSND
  • BSND Bartter syndrome IV
  • DRTA distal renal tubular acidosis
  • Usher syndrome (also known as Hallgren syndrome, Usher-Hallgren syndrome, retinitis pigmentosa-dysacusis syndrome, and dystrophia retinae dysacusis syndrome) is a rare disorder caused by a mutation in any one of at least ten genes, resulting in a combination of hearing loss and a gradual visual impairment, and is a leading cause of deafblindness.
  • the hearing loss is caused by a defective inner ear, whereas the vision loss results from retinitis pigmentosa (RP), a degeneration of the retinal cells.
  • Usher syndrome has three clinical subtypes, denoted as I, II, and III.
  • Subjects with Usher I are born profoundly deaf and begin to lose their vision in the first decade of life, learn to walk slowly as children due to problems in their vestibular system, and exhibit balance difficulties.
  • Subjects with Usher II are not born deaf, but do have hearing loss, but do not seem to have noticeable problems with balance; they also begin to lose their vision later (in the second decade of life) and may preserve some vision even into middle age.
  • Subjects with Usher syndrome III are not born deaf, but experience a gradual loss of their hearing and vision; they may or may not have balance difficulties.
  • VGLUT3 vesicular glutamate transporter-3
  • Wolfram syndrome is a condition that affects many of the body's systems, most often characterized by high blood sugar levels resulting from a shortage of the hormone insulin (diabetes mellitus) and progressive vision loss due to degeneration of the nerves that carry information from the eyes to the brain (optic atrophy).
  • people with Wolfram syndrome often also have pituitary gland dysfunction that results in the excretion of excessive amounts of urine (diabetes insipidus), hearing loss caused by changes in the inner ear (sensorineural deafness), urinary tract problems, reduced amounts of the sex hormone testosterone in males (hypogonadism), or neurological or psychiatric disorders.
  • the WFS1 gene encodes a protein called wolframin thought to regulate the amount of calcium in cells.
  • Wolfram syndrome is caused by mutations in the WFS1 gene, it is inherited in an autosomal recessive pattern, and the wolframin protein has reduced or absent function.
  • the endoplasmic reticulum does not work correctly.
  • the cell triggers its own cell death (apoptosis).
  • the death of cells in the pancreas, specifically cells that make insulin (beta cells) causes diabetes mellitus in people with Wolfram syndrome.
  • the gradual loss of cells along the optic nerve eventually leads to blindness in affected individuals.
  • the death of cells in other body systems likely causes the various signs and symptoms of Wolfram syndrome type 1.
  • Nonsyndromic mitochondrial hearing loss and deafness is characterized by moderate-to-profound hearing loss.
  • Pathogenic variants in MT-TS1 are usually associated with childhood onset of sensorineural hearing loss.
  • Pathogenic variants in MT-RNR1 are associated with predisposition to hearing loss if they are exposed to certain antibiotic medications called aminoglycosides (ototoxicity) and/or late-onset sensorineural hearing loss; however, some people with a mutation in the MT-RNR1 gene develop hearing loss even without exposure to these antibiotics.
  • Hearing loss associated with aminoglycoside ototoxicity is bilateral and severe to profound, occurring within a few days to weeks after administration of any amount (even a single dose) of an aminoglycoside antibiotic such as gentamycin, tobramycin, amikacin, kanamycin, or streptomycin.
  • an aminoglycoside antibiotic such as gentamycin, tobramycin, amikacin, kanamycin, or streptomycin.
  • ischemic damage may be prevented by compounds such as insulin-like growth factor (IGF-1), AM-111 (an apoptosis inhibitor), edarabone (a free radical scavenger), ginsenoside RB 1 (Kappo), glia-cell derived neurotrophic factor (GDNF), BDNF, CNTF, SOD1, SOD2, Necrostatin-1, DFNA5 and MSRB3.
  • IGF-1 insulin-like growth factor
  • AM-111 an apoptosis inhibitor
  • edarabone a free radical scavenger
  • ginsenoside RB 1 Kappo
  • GDNF glia-cell derived neurotrophic factor
  • BDNF BDNF
  • CNTF CNTF
  • SOD1, SOD2 Necrostatin-1
  • DFNA5 neurotrophic or hormonal control mechanisms
  • JNK-1 induced apoptosis may be prevented by compounds such as dominant-negative JNK-1 and d-steroisomer JNK-1 (Mol. Pharmacol. 2007 March; 71(3):654-66; the contents of which are herein incorporated by reference in its entirety).
  • FIG 1 is an exemplary schematic representation of a genetic map of the 5’ and 3’ vectors for dual-AAV transduction in inner hair cells (IHCs) using the trans-splicing approach.
  • a CMV enhancer (CMVe) and a human b-actin promoter (hbA) drive the transcription of an mRNA coding for eGFP and a P2A peptide, which is cleaved during translation.
  • the 5’ vector also contains cDNA encoding an N-terminal portion of otoferlin and a splice donor site (SD). The SD DNA sequence was provided by Trapani et al . (2014) EMBO Mol Med 6 194-211.
  • SA splice acceptor site
  • ITR first inverted terminal repeat
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • pA poly-adenylation signal
  • Figure 2 is an exemplary schematic representation of a genetic map of the 5’ and 3’ vectors for dual-AAV transduction in IHCs, using the hybrid approach.
  • highly recombinogenic sequences from FI phage (AK) sequences were included at the 3’ end of the ‘5 vector and at the 5’ end of the 3’ vector to force correct vector assembly (Trapani et al. (2014) EMBO Mol Med 6 194-211).
  • ABR auditory brainstem responses
  • eGFP enhanced green fluorescent protein
  • Figure 5 is an exemplary graph showing ABR threshold (in dB SPL) over frequency (in kHz) in response to pure tones or click stimuli.
  • the minimal sound pressure level (SPL) at which waves can be detected is displayed for individual Otof animals with dual-AAV mediated expression of otoferlin (white circles represent individual animals, circle with diagonal line fill is the average threshold across all animals), for non-transduced Otof animals (black circles), and for Oto animals after dual-AAV with eGFP transduction (circle with checkered fill), in response to pure tones or click stimuli.
  • Figure 6 is a graph showing the transduction rates of IHCs along the cochlea (e.g., entire Corti, apical turn, and midbasal/basal turn), determined by immunohistochemistry using two different antibodies, one binding the N-terminal otoferlin fragment, and the other binding to the very C-terminal part of otoferlin.
  • cochlea e.g., entire Corti, apical turn, and midbasal/basal turn
  • Figure 7 is a set of immunohistochemical images of one organ of Corti from an Otof mouse after dual-AAV mediated expression of otoferlin.
  • Calbindin was used as marker for inner and outer hair cells.
  • Cells expressing eGFP indicate virus transduction of at least the 5’ virus.
  • Left and middle panels show anti-otoferlin antibody staining (Abeam) in the N-terminal half of otoferlin.
  • Right panel shows C-terminal anti-otoferlin antibody staining (Synaptic Systems). Together, all three panels demonstrate that full- length otoferlin is expressed in IHCs.
  • AAVs transduce several cell types in the organ of Corti (indicated by eGFP fluorescence), otoferlin expression was restricted to inner hair cells.
  • Scale bar 100 pm.
  • ACm plasma membrane capacitance
  • Figure 10 is a representative graph showing Qreal over the duration of depolarization (ms) in Otof IHCs (white diamond), dual-AAV transduced IHCs of
  • Figure 11 is a representative plasmid map of pAAV-AK-SA-3’mOTOF-EWB.
  • Figure 12 is a representative plasmid map of pAAV-SA-3’mOTOF-EWB.
  • Figure 13 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5’mOTOF- SD.
  • Figure 14 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5’mOTOF- SD-AK.
  • Figure 15 is a graph showing plasma membrane capacitance (ACm) over the duration of depolarization in IHCs of Otof mice transduced with the two vectors shown in Figure 2 (medium shading; thick line), IHCs in wildtype mice (dark shading; medium thickness line), and IHCs in Otof mice.
  • ACm plasma membrane capacitance
  • Figure 16 is a representative graph showing Qreal over the duration of depolarization (ms) in Otof IHCs (white diamond), IHCs of Otof mice transduced with the two vectors shown in Figure 2 (gray diamonds), and IHCs of background- matched wild-type controls (black diamond).
  • Figure 17 is a representative plasmid map of pAAV-AK-SA-3’mOTOF-EWB.
  • Figure 18 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5’mOTOF- SD.
  • Figure 19 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5’mOTOF- SD-AK.
  • Figure 20 is a representative plasmid map of pAAV-SA-3’mOTOF-EWB.
  • Figure 21 is a representative schematic of a portion of pAKOS102 (SEQ ID NO: 43).
  • Figure 22 is a representative schematic of a portion of pAKOS103 (SEQ ID NO: 44).
  • Figure 23 is a representative schematic of a portion of pAKOS103.
  • Figure 24 is a representative plasmid map of pAKOS104 (SEQ ID NO: 45).
  • Figure 25 is a representative schematic of a portion of pAKOS104-DHFR (SEQ ID NO: 46).
  • Figure 26 is a representative schematic of a portion of pAKOS104-DHFR.
  • Figure 27 is a representative schematic of a portion of pAKOS105 (SEQ ID NO:
  • Figure 28 is a representative schematic of a portion of pAKOS105.
  • Figure 29 is a representative schematic of a portion of pAKOS105_GFP (SEQ ID NO: 48).
  • Figure 30 is a representative schematic of a portion of pAKOS106 (SEQ ID NO:
  • Figure 31 is a representative schematic of a portion of pAKOS106.
  • Figure 32 is a representative schematic of a portion of pAKOS107 (SEQ ID NO:
  • Figure 33 is a representative schematic of a portion of pAKOS107.
  • Figure 34 is a representative schematic of a portion of pAKOS108 (SEQ ID NO:
  • Figure 35 is a representative schematic of a portion of pAKOS108.
  • Figure 36 is a representative schematic of a portion of pAKOS109 (SEQ ID NO:
  • Figure 37 is a representative schematic a factor VIII stuffer (SEQ ID NOs. 54-57).
  • Figure 38 is a representative schematic of pl09 (SEQ ID NO: 84).
  • Figure 39 is a representative schematic of pl05 (SEQ ID NO: 85).
  • Figure 40 is a representative schematic of 105.WPRE.
  • Figure 41 is a representative schematic of pl08.
  • Figure 42 is a representative schematic of 10TOF18.CL1.
  • Figure 43 is a representative schematic of 190T0F48.
  • Figure 44 is a representative schematic of 1OTOF20.CL1.
  • Figure 45 is a representative schematic of 210T0F48.WPRE.
  • Figure 46 is a representative schematic of 10TOF21.CL1.
  • Figure 47 is a representative schematic of 220T0F48.WPRE.
  • Figure 48 is a representative schematic of 105.pA.NTF3.CMVd.
  • Figure 49 is an immunoblot showing the expression of full-length human otoferlin in HEK293FT cells transfected with the different pairs of plasmids indicated.
  • Figure 50 is a table showing the quantitation of expression of full-length human otoferlin from three replicates of the experiment described in Figure 49.
  • Figure 51 is a graph of the click ABR threshold in wildtype not treated with a vector or Otof mice not treated with a vector or treated with DualAAV Anc80.hOtof vectors (pi 05 and pi 09 vectors). Hearing in the Otof mice administered the DualAAV Anc80.hOtof vectors (pi 05 and 109 vectors) was measured at 26-28 days and 91 days after treatment.
  • Figure 52 is a graph of the tone burst ABR threshold in wildtype not treated with a vector or Otof mice not treated with a vector or treated with DualAAV Anc80.hOtof vectors (pi 05 and pi 09 vectors). Hearing in the Otof /_ mice administered the DualAAV Anc80.hOtof vectors (pi 05 and 109 vectors) was measured at 26-28 days and 91 days after treatment.
  • Figure 53 is a representative schematic of a portion of pAAV-HBA-eGFP-P2A- 5’mOTOF.SD (SEQ ID NO: 87).
  • Figure 54 is a representative schematic of a portion of pAAV-SA- 3’mOTOF.WPRE (SEQ ID NO: 88).
  • Figure 55 is a representative schematic of a portion of pAAV-HBA-eGFP-P2A- 5’mOTOF.SD-AK.
  • Figure 56 is a representative schematic of a portion of pAAV-AK-SA- 3’mOTOF.WPRE.
  • Figure 57 is a representative schematic of a portion of pAAV-CMV-5’hOTOF- SD-AK.
  • Figure 58 is a representative schematic of a portion of pAAV-HBA-5’hOTOF- SD-AP.
  • Figure 59 is a representative schematic of a portion of pAAV-HBA-5’hOTOF- SD-AK.
  • Figure 60 is a representative schematic of a portion of pAAV-HBA- 5 ’ hOTOF codop-SD-AK.
  • Figure 61 is a representative schematic of a portion of pAAV-HBA- 5 ’ hOTOF codop- SD .
  • Figure 62 is a representative schematic of a portion of pAAV-CMV- 5 ’ hOTOF codop- SD .
  • Figure 63 is a representative schematic of a portion of pAAV-CMV- 5 ’ hOTOF codop-SD-AK.
  • Figure 64 is a representative schematic of a portion of pAAV-CBA- 5 ’ hOTOF codop-SD-AK.
  • Figure 65 is a representative schematic of a portion of pAAV-CBA-5’hOTOF- SD.
  • Figure 66 is a representative schematic of a portion of pAAV-SA-3OTOF.
  • Figure 67 is a representative schematic of a portion of pAAV-AP-SA-3OTOF.
  • Figure 68 is a representative schematic of a portion of pAAV-AK-SA- 3’OTOFcodop.
  • Figure 69 is a representative immunoblot showing the expression of full-length human otoferlin in HEK293FT cells transfected using DNA transfection reagent jetPRIME® (polyplus) with 600 ng of the different pairs of plasmids indicated.
  • Lane 1 contained a prestained protein ladder.
  • Lane 2 contained a protein sample of HEK293FT cells that were transfected with vector pAKOS104 (as shown in Figures 24 and 59) and vector pAKOS105 (as shown in Figures 27, 28 and 39).
  • Lane 3 contained a protein sample of HEK293FT cells that were transfected with vector pAKOS108 (as shown in Figures 34, 35, 41 and 57) and vector pAKOS105 (as shown in Figures 27, 28 and 39).
  • Lane 4 contained a protein sample of HEK293FT cells that were transfected with vector pAKOS109 (as shown in Figures 36 and 38) and vector pAKOS105 (as shown in Figures 27, 28 and 39).
  • Lane 5 contained a protein sample of HEK293FT cells that were transfected with vector pAAV-HBA-5’hOTOFcodop-SD-AK (as shown in Figure 60) and vector pAAV-AK-SA-3’OTOFcodop (as shown in Figure 68).
  • Lane 6 contained a protein sample of HEK293FT cells that were transfected with vector pAAV-CMV- 5’ hOTOF codop-SD-AK (as shown in Figure 63) and vector pAAV-AK-SA- 3’OTOFcodop (as shown in Figure 68).
  • Lane 7 contained a protein sample of HEK293FT cells that were transfected with vector pAAV_CBA-5’hOTOFcodop-SD-AK (as shown in Figure 64) and vector pAAV-AK-SA-3’OTOFcodop (as shown in Figure 68).
  • Lane 8 contained a protein sample of HEK293FT cells that were transfected with vector pAKOS102 (as shown in Figure 21) and vector pAKOS103 (as shown in Figures 22, 23 and 66).
  • Lane 9 contained a protein sample of HEK293FT cells that were transfected with a CBA.TS vector and vector pAKOS103 (as shown in Figures 22, 23 and 66).
  • Lane 10 contained a protein sample of HEK293FT cells that were transfected with vector pAKOS106 (as shown in Figures 30, 31 and 58) and pAKOS107 (as shown in Figures 32, 33 and 67).
  • pAKOS106 as shown in Figures 30, 31 and 58
  • pAKOS107 as shown in Figures 32, 33 and 67.
  • RIPA buffer analyzed in 4-12% Bolt protein gel, which was then transferred onto a nitrocellulose membrane.
  • Human otoferlin was detected using an anti- OTOF polyclonal antibody (Thermo PA5-52935).
  • Human beta-actin was used as the primary antibody for internal loading control between lanes.
  • the experiment was repeated in triplicate. Relative quantitative measurements for each experiment are provided under the immunoblot, along with the average measurement and standard deviation (STDEV).
  • Figure 70 is an immunoblot showing the expression of full-length human otoferlin in HEK293FT cells transfected with the different pairs of plasmids indicated at different multiplicity of infections (MOI).
  • HEK293FT cells were seeded overnight at 4 x 10 4 cells/well on a 96-well plate. Six hours post-seeding, the dual vectors were added to each well. Ninety-six hours post-transfection, cells were harvested and lysed using RIPA buffer and analyzed in 4-12% Bolt protein gel, which was then transferred onto a nitrocellulose membrane. Human otoferlin was detected using an anti-OTOF polyclonal antibody (Thermo PA5-52935).
  • Lane 1 CBA.OTOF(AK) with MOI 503,000; lane 2: CBA.OTOF(AK) with MOI 1,510,000; lane 3: CBA.OTOF(AK) with MOI 100,000; lane 4: CBA.OTOF(AK) with MOI 303,000; lane 5: CMV.OTOF(AK) with MOI 638,000; lane 6: CMV.OTOF(AK) with MOI 1,910,000; lane 7: CMV.OTOF(AK) with MOI 127,000; lane 8: CMV.OTOF(AK) with MOI 382,000; lane 9: negative control.
  • Figure 71 a set of immunohistochemical images of one organ of Corti from an Otof-/- mouse age P17 after unilateral intracochlear administration of dual-AAV vectors expressing CBA.hOTOF(AK) (pl05 and 109 vectors). Ipsilateral cochlea was dissected and analyzed for protein expressing using immunohistochemistry at three different frequency regions (base - apex).
  • IHC N- and C- terminal otoferlin labeled inner hair cells
  • Figure 73 is a graph showing the average N-terminal and C-terminal otoferlin immunofluorescence levels in dual-AAV-transduced Otof and wild-type inner hair cells (IHC) from mice (aged P23-30). Otoferlin levels were normalized to immunofluorescence levels in non-transduced B6 wild-type IHCs for each antibody separately. The number of quantified IHCs is indicated inside the bars. Data are displayed as mean ⁇ standard error of mean (s.e.m.), ns P>0.05; *P ⁇ 0.05; **P ⁇ 0.01; ***p ⁇ 0.001, Kruskal-Wallis test followed by Dunn’s multiple comparison test.
  • Figure 76 is a graph showing summed auditory brainstem response (ABR) wave I-V amplitudes at different click sound intensities in otoferlin dual-AAV-injected, non- injected Otof ⁇ , and wild-type control mice aged P23-30.
  • n 38 mice). Data are represented as mean ⁇ standard error of mean (s.e.m.) Individual animals are depicted with open symbols.
  • ABR summer auditory brainstem response
  • IHC inner hair cell
  • Figure 78 is a schematic of an exemplary dual AAV vector system of the present disclosure which includes “upstream” and “downstream” vectors (AKhOTOF5 and AKhOTOF3, respectively).
  • Figure 79 is a schematic of the “upstream” vector AKhOTOF5.
  • Figure 80 is a schematic of the “downstream” vector AKhOTOF3.
  • Figure 81 illustrates a perspective of a device for delivering fluid to an inner ear, according to aspects of the present disclosure.
  • Figure 82 illustrates a sideview of a bent needle sub-assembly, according to aspects of the present disclosure.
  • Figure 83 illustrates a perspective view of a device for delivering fluid to an inner ear, according to aspects of the present disclosure.
  • Figure 84 illustrates a perspective view of a bent needle sub-assembly coupled to the distal end of a device, according to aspects of the present disclosure.
  • compositions including at least two different nucleic acid vectors, where each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein can be used to generate a sequence encoding an active otoferlin protein (e.g., a full-length otoferlin protein) in a mammalian cell, and thereby treat non-syndromic sensorineural hearing loss in a subject in need thereof.
  • active otoferlin protein e.g., a full-length otoferlin protein
  • compositions that include at least two different nucleic acid vectors, wherein: each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, each of the encoded portions being at least 30 amino acid residues in length, wherein the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes a full-length otoferlin protein; at least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons; and when introduced into a mammalian cell the at least two different vectors undergo concatemerization or homologous recombination with each other, thereby forming a recombined nucleic acid that encodes a full-length otoferlin protein.
  • each of the at least two different vectors is a plasmid, a transposon, a cosmid, an artificial chromosome, or a viral vector.
  • each of the at least two different vectors is a human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or a PI -derived artificial chromosome (PAC).
  • each of the at least two different vectors is a viral vector selected from an adeno- associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, or a retrovirus vector. In some embodiments of any of the compositions described herein, each of the at least two different vectors is an AAV vector.
  • AAV adeno- associated virus
  • the amino acid sequence of one of the encoded portions overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments of any of the compositions described herein, the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different encoded portion. In some embodiments of any of the compositions described herein, the overlapping amino acid sequence is between about 30 amino acid residues to about 1000 amino acid residues in length.
  • the vectors include two different vectors, each of which includes a different segment of an intron, wherein the intron includes the nucleotide sequence of an intron that is present in otoferlin genomic DNA, and wherein the two different segments overlap in sequence by at least 100 nucleotides. In some embodiments of any of the compositions described herein, the two different segments overlap in sequence by about 100 nucleotides to about 800 nucleotides. In some embodiments of any of the compositions described herein, the nucleotide sequence of each of the at least two different vectors is between about 500 nucleotides to about 10,000 nucleotides in length. In some embodiments of any of the compositions described herein, the nucleotide sequence of each of the at least two different vectors is between 500 nucleotides to 5,000 nucleotides in length.
  • the number of different vectors in the composition is two.
  • a first of the two different vectors includes a coding sequence that encodes an N-terminal portion of the otoferlin protein.
  • the N-terminal portion of the otoferlin protein is between 30 amino acids to 1600 amino acids in length.
  • the N-terminal portion of the otoferlin protein is between 200 amino acids to 1500 amino acids in length.
  • the first vector further includes one or both of a promoter and a Kozak sequence.
  • the first vector includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.
  • one of the two vectors comprises SEQ ID NO: 39 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 39) and the second of the two vectors comprises SEQ ID NO: 40 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 40).
  • one of the two vectors comprises SEQ ID NO: 41 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 41) and the second of the two vectors comprises SEQ ID NO: 42 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 42).
  • one of the two vectors comprises SEQ ID NO:84 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 84) and the second of the two vectors comprises SEQ ID NO: 85 (or comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 85).
  • one of the at least two different vectors comprises a sequence encoding a NTF3 protein.
  • compositions described herein wherein the sequence encoding a NTF3 protein is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 78.
  • the first vector further includes a coding sequence encoding a destabilization domain, wherein the destabilization domain is 3’ to the coding sequence that encodes the N-terminal portion of the otoferlin protein.
  • the coding sequence that encodes the N-terminal portion of the otoferlin protein comprises exons 1- 21 of isoform 5 of the human otoferlin gene.
  • the second of the two different vectors includes a coding sequence that encodes a C-terminal portion of the otoferlin protein.
  • the C-terminal portion of the otoferlin protein is between 30 amino acids to 1600 amino acids in length. In some embodiments of any of the compositions described herein, the C-terminal portion of the otoferlin protein is between 200 amino acids to 1500 amino acids in length.
  • the second vector further includes a poly(dA) signal sequence.
  • the coding sequence that encodes the C-terminal portion of the otoferlin protein comprises exons 22-48 of isoform 5 of the human otoferlin gene.
  • the second vector further includes sequences for mRNA stabilization. Some embodiments of any of the compositions described herein further include a pharmaceutically acceptable excipient.
  • kits that include any of the compositions described herein. Some embodiments of any of the kits described herein further include a pre- loaded syringe containing the composition.
  • kits that include introducing into a cochlea of a mammal a therapeutically effective amount of any of the compositions described herein.
  • the mammal is a human.
  • the mammal has been previously identified as having a defective otoferlin gene.
  • Also provided herein are methods of increasing expression of an active otoferlin protein, e.g., a full-length otoferlin protein, in a mammalian cell that include introducing any of the compositions described herein into the mammalian cell.
  • the mammalian cell is a cochlear inner hair cell.
  • the mammalian cell is a human cell.
  • the mammalian cell has previously been determined to have a defective otoferlin gene.
  • Also provided herein are methods of increasing expression of an active otoferlin protein, e.g., a full-length otoferlin protein in an inner hair cell in a cochlea of a mammal that include: introducing into the cochlea of the mammal a therapeutically effective amount of any of the compositions described herein.
  • the mammal has been previously identified as having a defective otoferlin gene.
  • the mammal is a human.
  • the subject is a human.
  • Some embodiments of any of the methods described herein further include, prior to the administering step, determining that the subject has a defective otoferlin gene.
  • compositions that include two different nucleic acid vectors, wherein: a first nucleic acid vector of the two different nucleic acid vectors includes a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin protein positioned 3’ of the promoter, and a splicing donor signal sequence positioned at the 3’ end of the first coding sequence; and a second nucleic acid vector of the two different nucleic acid vectors includes a splicing acceptor signal sequence, a second coding sequence that encodes a C-terminal portion of an otoferlin protein positioned at the 3’ end of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3 ’ end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the amino acid sequences of the encoded portions do not overlap, wherein no single vector of the two different vectors encodes a full-length otofer
  • the coding sequence of at least one of the vectors includes a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons.
  • compositions that include: a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin protein positioned 3’ of the promoter, a splicing donor signal sequence positioned at the 3’ end of the first coding sequence, and a first detectable marker gene positioned 3’ of the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second detectable marker gene, a splicing acceptor signal sequence positioned 3’ of the second detectable marker gene, a second coding sequence that encodes a C-terminal portion of an otoferlin protein positioned at the 3’ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3’ end of the second coding sequence; wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the respective amino acid sequences of the encoded portions do not overlap with
  • the coding sequence of at least one of the vectors includes a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons.
  • the first or second detectable marker gene encodes alkaline phosphatase. In some embodiments of any of the compositions described herein, the first and second detectable marker genes are the same.
  • compositions that include a first nucleic acid vector including a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin protein positioned 3’ to the promoter, a splicing donor signal sequence positioned at the 3’ end of the first coding sequence, and a highly recombinogenic sequence (e.g., a FI phage recombinogenic region, e.g., SEQ ID NO: 66) positioned 3’ to the splicing donor signal sequence; and a second nucleic acid vector, different from the first nucleic acid vector, including a second highly recombinogenic sequence (e.g., a FI phage recombinogenic region, e.g., SEQ ID NO: 67, or an alkaline phosphatase recombinogenic region, e.g., SEQ ID NO: 89), a splicing acceptor signal sequence positioned 3’ of the second highly recombin
  • the coding sequence of at least one of the vectors includes a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons.
  • kits that include any of the compositions described herein. Some embodiments of any of the kits described herein further include a pre- loaded syringe containing the composition.
  • kits that include introducing into a cochlea of a mammal a therapeutically effective amount of any of the compositions described herein.
  • the mammal is a human.
  • the mammal has been previously identified as having a defective otoferlin gene.
  • kits for increasing expression of a full-length otoferlin protein in a mammalian cell that include introducing any of the compositions described herein into the mammalian cell.
  • the mammalian cell is a cochlear inner hair cell.
  • the mammalian cell is a human cell.
  • the mammalian cell has previously been determined to have a defective otoferlin gene.
  • Also provided herein are methods of increasing expression of a full-length otoferlin protein in an inner hair cell in a cochlea of a mammal that include introducing into the cochlea a therapeutically effective amount of any of the compositions described herein.
  • the mammal has been previously identified as having a defective otoferlin gene.
  • the mammal is a human.
  • Also provided herein are methods of treating non-symptomatic sensorineural hearing loss in a subject identified as having a defective otoferlin gene that include administering a therapeutically effective amount of any of the compositions described herein into a cochlea of the subject.
  • the subject is a human.
  • Some embodiments of any of the methods described herein further include, prior to the administering step, determining that the subject has a defective otoferlin gene.
  • compositions including a plurality of adeno- associated viral (AAV) vectors, wherein the plurality of AAV vectors are capable of constituting an auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • AAV adeno- associated viral
  • the plurality of AAV vectors are capable of constituting a full-length auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • Some embodiments of any of the therapeutic composition described herein further include a first AAV vector and a second AAV vector, wherein the first and second AAV vectors independently contain packaging capacity of less than about 6kb.
  • the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group of otoferlin and an ortholog or homolog thereof.
  • any of the therapeutic compositions described herein can further include a nucleic acid (e.g., a vector) including a nucleic acid sequence encoding an auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group consisting of Ca v 1.3, a scaffold protein selected from bassoon, piccolo, ribeye, and harmonin, Vglut3, synaptotagmin, a vesicle tethering / docking protein, a vesicle priming protein, a vesicle fusion proteins, GluA2/3, and GluA4.
  • the first AAV vector further includes at least one promoter sequence selected from a CBA, a CMV, or a CB7 promoter.
  • the first AAV vector further includes at least one promoter sequence selected from Cochlea-specific promoters.
  • the therapeutic composition is formulated for intra-cochlear administration. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include a lipid nanoparticle. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include a polymeric nanoparticle. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include a mini-circle DNA. In some embodiments of any of the therapeutic compositions described herein, the therapeutic composition is formulated to include a CELiD DNA. In some embodiments of any of the compositions described herein, the therapeutic composition is formulated to include a synthetic perilymph solution.
  • the therapeutic composition is formulated to include a synthetic perilymph solution including 20-200mM NaCl; 1-5 mM KC1; O.l-lOmM CaCl2; 1-lOmM glucose; and 2-50 mM HEPES; and having a pH of between about 6 and about 9.
  • compositions that include an auditory polypeptide messenger RNA.
  • compositions that include one or a plurality of adenoviral (AV) vectors, where the one or the plurality of AV vectors are capable of constituting an auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • AV adenoviral
  • the one or the plurality of AV vectors are capable of constituting a full-length auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • therapeutic compositions including one or a plurality of lentiviral vectors, where the one or the plurality of lentiviral vectors are capable of constituting an auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • the one or the plurality of lentiviral vectors are capable of constituting an active, e.g., full- length auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • surgical methods that include the steps of: i) introducing into a cochlea of a human subject a first incision at a first incision point; ii) administering intra-cochlearly an effective dose of a therapeutic composition (e.g., any of the therapeutic compositions described herein).
  • a therapeutic composition e.g., any of the therapeutic compositions described herein.
  • the therapeutic composition is administered to the subject at the first incision point. In some embodiments of any of the methods described herein, the therapeutic composition is administered to the subject into or through the first incision.
  • the therapeutic composition is administered to the subject into or through the cochlea oval window membrane. In some embodiments of any of the methods described herein, the therapeutic composition is administered to the subject into or through the cochlea round window membrane.
  • the therapeutic composition is administered using a medical device capable of creating a plurality of incisions in the round window membrane.
  • the medical device includes a plurality of micro-needles. In some embodiments of any of the methods described herein, the medical device includes a plurality of micro-needles including a generally circular first aspect, where each micro-needle has a diameter of at least about 10 microns.
  • the medical device includes a base and/or a reservoir capable of holding the therapeutic composition. In some embodiments of any of the methods described herein, the medical device includes a plurality of hollow micro-needles individually including a lumen capable of transferring the therapeutic composition.
  • the medical device includes a means for generating at least a partial vacuum.
  • therapeutic delivery systems that include i) a medical device capable of creating a plurality of incisions in a round window membrane of an inner ear of a human subject in need thereof, and ii) an effective dose of a therapeutic composition including a plurality of adeno-associated viral (AAV) vectors, wherein the plurality of AAV vectors are capable of constituting an active, e.g., full-length, auditory polypeptide messenger RNA in a target cell of the inner ear.
  • AAV adeno-associated viral
  • Also provided herein are means for performing a surgical method that includes the steps of: i) administering intra-cochlearly to a human subject in need thereof an effective dose of the therapeutic composition (e.g., any of the therapeutic composition described herein), where the therapeutic composition is capable of being administered by using a medical device including a) means for creating a plurality of incisions in the round window membrane and b) the effective dose of the therapeutic composition.
  • an effective dose of the therapeutic composition e.g., any of the therapeutic composition described herein
  • the medical device includes a plurality of micro-needles.
  • compositions that include a single adeno- associated viral (AAV) vector, where the AAV vector is capable of constituting an auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • AAV adeno- associated viral
  • the single AAV vector is capable of constituting a full-length auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • a single vector e.g., any of the vectors described herein
  • that includes a sequence encoding an active otoferlin protein e.g., any of the full-length or truncated active otoferlin proteins described herein
  • an active otoferlin protein e.g., any of the full-length or truncated active otoferlin proteins described herein
  • the therapeutic composition further includes a second vector other than an AAV vector, where the single AAV vector and the second vector independently contain packaging capacity of less than about 6kb.
  • the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group of otoferlin and an ortholog or homolog thereof.
  • the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group of otoferlin and truncation mutant thereof.
  • the otoferlin truncation mutant includes at least a single C2 domain of the following:
  • the otoferlin truncation mutant does not include an endogenous otoferlin polypeptide C- terminal region.
  • the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group consisting of Ca v 1.3, a scaffold protein selected from bassoon, piccolo, ribeye, and harmonin, Vglut3, synaptotagmin, a vesicle tethering / docking protein, a vesicle priming protein, a vesicle fusion proteins, GluA2/3, and GluA4.
  • the single AAV vector further includes at least one promoter sequence selected from a CBA, a CMV, or a CB7 promoter.
  • the single AAV vector further includes at least one promoter sequence selected from a Cochlea-specific promoters.
  • the therapeutic composition is formulated for intra-cochlear administration.
  • the therapeutic composition is formulated to include a lipid nanoparticle.
  • the therapeutic composition is formulated to include a polymeric nanoparticle.
  • the therapeutic composition is formulated to include a mini-circle DNA.
  • the therapeutic composition is formulated to include a CELiD DNA.
  • the therapeutic composition is formulated to include a synthetic perilymph solution.
  • the therapeutic composition is formulated to include a synthetic perilymph solution including 20-200mM NaCl; 1-5 mM KCl; O.l-lOmM CaCl2; 1-lOmM glucose; 2-50 mM HEPES; having a pH of between about 6 and about 9.
  • compositions that include an auditory polypeptide messenger RNA encoding an otoferlin truncation mutant.
  • an element refers to one element and more than one element.
  • signaling domain refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • Otoferlin is believed to be a calcium sensor required for exocytosis in inner hair cells, as well as neurotransmitter release in immature outer hair cells.
  • otoferlin In the presence of phosphatidylserine (PS), calcium concentrations of 10 pM result in significant C2- liposome interaction for the C2C-C2E domains of otoferlin.
  • PS phosphatidylserine
  • otoferlin possesses domains that appear to operate using an “electrostatic switch” mechanism, as well as domains that bind regardless of calcium.
  • PI(4,5)P2 a major signaling molecule at the presynapse, has been shown to interact with the C2C and C2F domains of otoferlin in a calcium-independent fashion (Padmanarayana et al. 2014 Biochem 53:5023-5033).
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule, which specifically binds with an antigen.
  • Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.
  • antibody fragment refers to at least one portion of an intact antibody, or recombinant variants thereof, and refers to the antigen binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.
  • scFv refers to a fusion protein including at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • recombinant polypeptide refers to a polypeptide which is generated using recombinant DNA technology, such as, for example, a polypeptide expressed by a viral vector expression system.
  • the term should also be construed to mean a polypeptide which has been generated by the synthesis of a DNA molecule encoding the polypeptide and which DNA molecule expresses a protein, or an amino acid sequence specifying the polypeptide, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • mutation in an otoferlin gene refers to a modification in a wildtype otoferlin gene that results in the production of an otoferlin protein having one or more of: a deletion of one or more amino acids, one or more amino acid substitutions, and one or more amino acid insertions, as compared to the wildtype otoferlin protein, and/or results in a decrease in the expressed level of the encoded otoferlin protein in a mammalian cell as compared to the expressed level of the encoded otoferlin protein in a mammalian cell not having the mutation.
  • a mutation can result in the production of an otoferlin protein having a deletion of one or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, or 20 amino acids).
  • the mutation can result in a frameshift in the otoferlin gene.
  • the term “frameshift” is known in the art to encompass any mutation in a coding sequence that results in a shift in the reading frame of the coding sequence.
  • a frameshift can result in a nonfunctional protein.
  • a point mutation can be a nonsense mutation (i.e., result in a premature stop codon in an exon of the gene).
  • a nonsense mutation can result in the production of a truncated protein (as compared to a corresponding wildtype protein) that may or may not be functional.
  • the mutation can result in the loss (or a decrease in the level) of expression of otoferlin mRNA or otoferlin protein or both the mRNA and protein.
  • the mutation can result in the production of an altered otoferlin protein having a loss or decrease in one or more biological activities (functions) as compared to a wildtype otoferlin protein.
  • the mutation is an insertion of one or more nucleotides into an otoferlin gene.
  • the mutation is in a regulatory sequence of the otoferlin gene, i.e., a portion of the gene that is not coding sequence.
  • a mutation in a regulatory sequence may be in a promoter or enhancer region and prevent or reduce the proper transcription of the otoferlin gene.
  • Modifications can be introduced into a nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antibody fragment of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid and glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine
  • beta-branched side chains e.g., threonine, valine, and isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, and histidine
  • encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of a defined sequence of amino acids, in accordance with the genetic code.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene, cDNA or RNA produces the protein.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription, can be referred to as encoding the protein product.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as two DNA molecules or two RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.
  • a nucleotide sequence that encodes a protein may also include introns.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from outside or produced outside an organism, cell, tissue or system.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • substantially purified cell refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cells that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
  • transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • the term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).
  • the subject is a rodent (e.g., a rat or mouse), a rabbit, a sheep, a dog, a cat, a horse, a non-human primate, or a human.
  • the subject has or is at risk of developing non- syndromic deafness.
  • the subject has been previously identified as having a mutation in an otoferlin gene.
  • the subject has been identified as having a mutation in an otoferlin gene and has been diagnosed with non- symptomatic sensorineural hearing loss.
  • the subject has been identified as having non- symptomatic sensorineural hearing loss.
  • therapeutic means a treatment.
  • a therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
  • prophylaxis means the prevention of, or protective treatment for, a disease or disease state. “Prevention” in this context includes reducing the likelihood the subject will experience the disease.
  • a therapeutically effective amount of a composition can result in an increase in the expression level of an active otoferlin protein (e.g., a wildtype, full-length otoferlin protein or of a variant of an otoferlin protein that has the desired activity) (e.g., as compared to the expression level prior to treatment with the composition).
  • an active otoferlin protein e.g., a wildtype, full-length otoferlin protein or of a variant of an otoferlin protein that has the desired activity
  • a therapeutically effective amount of a composition can result in an increase in the expression level of an active otoferlin protein (e.g., a wildtype, full-length otoferlin protein or active variant) in a target cell (e.g., a cochlear inner hair cell).
  • a therapeutically effective amount of a composition can result in a different cellular localization of an active otoferlin protein (e.g., a wildtype, full-length otoferlin protein or an active variant) in a target cell (e.g., a cochlear inner hair cell).
  • a therapeutically effective amount of a composition can result in an increase in the expression level of an active otoferlin protein (e.g., a wildtype, full-length otoferlin protein or active variant), and/or an increase in one or more activities of an otoferlin protein in a target cell (e.g., as compared to a reference level, such as the level(s) in a subject prior to treatment, the level(s) in a subject having a mutation in an otoferlin gene, or the level(s) in a subject or a population of subjects having non- symptomatic sensorineural hearing loss).
  • an active otoferlin protein e.g., a wildtype, full-length otoferlin protein or active variant
  • an increase in one or more activities of an otoferlin protein in a target cell e.g., as compared to a reference level, such as the level(s) in a subject prior to treatment, the level(s) in
  • parenteral administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • in vitro transcribed RNA refers to RNA, preferably mRNA, that has been synthesized in vitro.
  • the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector includes a template that is used to generate the in vitro transcribed RNA.
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein including two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
  • active otoferlin protein means a protein encoded by DNA that, if substituted for both wildtype alleles encoding full-length otoferlin protein in auditory hair cells (e.g., auditory inner hair cells) of what is otherwise a wildtype mammal, and if expressed in the auditory hair cells of that mammal, results in that mammal’s having a level of hearing approximating the normal level of hearing of a similar mammal that is entirely wildtype.
  • active otoferlin proteins are full-length otoferlin proteins (e.g., any of the full-length otoferlin proteins described herein).
  • an active otoferlin protein can include a sequence of a wildtype, full-length otoferlin protein (e.g., a wildtype, human, full-length otoferlin protein) including 1 amino acid substitution to about 240 amino acid substitutions, 1 amino acid substitution to about 235 amino acid substitutions, 1 amino acid substitution to about 230 amino acid substitutions, 1 amino acid substitution to about 225 amino acid substitutions, 1 amino acid substitution to about 220 amino acid substitutions, 1 amino acid substitution to about 215 amino acid substitutions, 1 amino acid substitution to about 210 amino acid substitutions, 1 amino acid substitution to about 205 amino acid substitutions, 1 amino acid substitution to about 200 amino acid substitutions, 1 amino acid substitution to about 195 amino acid substitutions, 1 amino acid substitution to about 190 amino acid substitutions, 1 amino acid substitution to about 185 amino acid substitutions, 1 amino acid substitution to about 180 amino acid substitutions, 1 amino acid substitution to about 175 amino acid substitutions, 1 amino acid substitution to about 170 amino acid substitutions, 1 amino acid substitutions,
  • 110 amino acid substitutions to about 170 amino acid substitutions about 110 amino acid substitutions to about 165 amino acid substitutions, about 110 amino acid substitutions to about 160 amino acid substitutions, about 110 amino acid substitutions to about
  • 120 amino acid substitutions about 110 amino acid substitutions to about 115 amino acid substitutions, between about 120 amino acid substitutions to about 240 amino acid substitutions, about 120 amino acid substitutions to about 235 amino acid substitutions, about 120 amino acid substitutions to about 230 amino acid substitutions, about
  • 170 amino acid substitutions about 100 amino acid substitutions to about 165 amino acid substitutions, about 120 amino acid substitutions to about 160 amino acid substitutions, about 120 amino acid substitutions to about 155 amino acid substitutions, about
  • amino acids that are not conserved between wildtype otoferlin proteins from different species can be mutated without losing activity, while those amino acids that are conserved between wildtype otoferlin proteins from different species should not be mutated as they are more likely (than amino acids that are not conserved between different species) to be involved in activity.
  • An active otoferlin protein can include, e.g., a sequence of a wildtype, full-length otoferlin protein (e.g., a wildtype, human, full-length otoferlin protein) that has 1 amino acid to about 200 amino acids, 1 amino acid to about 195 amino acids, 1 amino acid to about 190 amino acids, 1 amino acid to about 185 amino acids, 1 amino acid to about 180 amino acids, 1 amino acid to about 175 amino acids, 1 amino acid to about 170 amino acids, 1 amino acid to about 165 amino acids, 1 amino acid to about 160 amino acids, 1 amino acid to about 155 amino acids, 1 amino acid to about 150 amino acids,
  • a wildtype, full-length otoferlin protein e.g., a wildtype, human, full-length otoferlin protein
  • amino acid to about 10 amino acids 1 amino acid to about 9 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 7 amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 5 amino acids, 1 amino acid to about 4 amino acids, 1 amino acid to about 3 amino acids, about 2 amino acids to about 200 amino acids, about
  • the two or more deleted amino acids can be contiguous in the sequence of the wildtype, full-length protein. In other examples where two or more amino acids are deleted from the sequence of a wildtype, full-length otoferlin protein, the two or more deleted amino acids are not contiguous in the sequence of the wildtype, full-length protein.
  • amino acids that are not conserved between wildtype, full-length otoferlin proteins from different species can be deleted without losing activity, while those amino acids that are conserved between wildtype, full-length otoferlin proteins from different species should not be deleted as they are more likely (than amino acids that are not conserved between different species) to be involved in activity.
  • an active otoferlin protein can, e.g., include a sequence of a wildtype, full-length otoferlin protein that has between 1 amino acid to about 100 amino acids, 1 amino acid to about 95 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 85 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 75 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid to about 65 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about 55 amino acids, 1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about 15 amino acids, 1 amino acid to about 10 amino acids,
  • 2 amino acids to about 25 amino acids about 2 amino acids to about 20 amino acids, about 2 amino acids to about 15 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 9 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 7 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 3 amino acids to about 100 amino acids, about 3 amino acid to about 95 amino acids, about 3 amino acids to about 90 amino acids, about 3 amino acids to about 85 amino acids, about 3 amino acids to about 80 amino acids, about 3 amino acids to about 75 amino acids, about 3 amino acids to about 70 amino acids, about 3 amino acids to about 65 amino acids, about 3 amino acids to about 60 amino acids, about 3 amino acids to about 55 amino acids, about 3 amino acids to about 50 amino acids, about 3 amino acids to about 45 amino acids, about 3 amino acids to about 40 amino acids, about 3 amino acids to about 100 amino acids, about 3 amino acid to about 95 amino acids, about 3 amino acids to about
  • 8 amino acids to about 90 amino acids about 8 amino acids to about 85 amino acids, about 8 amino acids to about 80 amino acids, about 8 amino acids to about 75 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 65 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 55 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acid to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 75 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 65 amino acids, about 10 amino acids to about
  • an active otoferlin protein can, e.g., include the sequence of a wildtype, full-length otoferlin protein where 1 amino acid to 50 amino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 35 amino acids, 1 amino acid to 30 amino acids, 1 amino acid to 25 amino acids, 1 amino acid to 20 amino acids, 1 amino acid to 15 amino acids, 1 amino acid to 10 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 6 amino acids, 1 amino acid to 5 amino acids, 1 amino acid to 4 amino acids, 1 amino acid to 3 amino acids, about 2 amino acids to 50 amino acids, about 2 amino acids to 45 amino acids, about 2 amino acids to 40 amino acids, about 2 amino acids to 35 amino acids, about 2 amino acids to 30 amino acids, about 2 amino acids to 25 amino acids, about 2 amino acids to 20 amino acids, about 2 amino acids to 15 amino acids, about 2 amino acids to 10 amino acids, about 2 amino acids
  • the inserted amino acid(s) can be inserted as a contiguous sequence into the sequence of a wildtype, full-length protein. In some examples, the amino acid(s) are not inserted as a contiguous sequence into the sequence of a wildtype, full-length protein. As can be appreciated in the art, the amino acid(s) can be inserted into a portion of the sequence of a wildtype, full-length protein that is not well-conserved between species.
  • ranges throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
  • the present disclosure provides a recombinant AAV vector of SEQ ID NO: 96. In one aspect the present disclosure provides a recombinant AAV vector of SEQ ID NO: 105.
  • the present disclosure provides a recombinant AAV vector that comprises, in order of 5’ to 3’ : a 5’ ITR sequence of SEQ ID NO: 97; a CAG promoter comprising a CMV early enhancer element of SEQ ID NO: 98, a chicken beta actin gene sequence of SEQ ID NO: 99, and a chimeric intron of SEQ ID NO: 100; a 5’OTOF coding region that comprises exons 1 to (and through) 21 of OTOF cDNA; a SD intron sequence of SEQ ID NO: 102; an AK recombinogenic sequence of SEQ ID NO: 103; and a 3’ ITR sequence of SEQ ID NO: 104.
  • the 5’OTOF coding region is SEQ ID NO: 101.
  • the 5OTOF coding region is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% identical to SEQ ID NO: 101, and encodes the same amino acid sequence as encoded by SEQ ID NO : 101.
  • the present disclosure provides a recombinant AAV vector that comprises, in order of 5’ to 3’ : a 5’ ITR sequence of SEQ ID NO: 97; an AK recombinogenic sequence of SEQ ID NO: 103; a SA intron sequence of SEQ ID NO:
  • the 3’OTOF coding region is SEQ ID NO:
  • the 3OTOF coding region is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% identical to SEQ ID NO: 107, and encodes the same amino acid sequence as encoded by SEQ ID NO: 107.
  • the present disclosure provides an rAAV particle comprising one of the aforementioned recombinant AAV vectors encapsidated by an Anc80 capsid.
  • the Anc80 capsid comprises a polypeptide of SEQ ID NO: 109.
  • the present disclosure provides a composition comprising a first rAAV particle comprising a recombinant AAV vector of SEQ ID NO: 96 and a second rAAV particle comprising a recombinant AAV vector of SEQ ID NO: 105.
  • the recombinant AAV vector of the first rAAV particle is encapsidated by an Anc80 capsid.
  • the recombinant AAV vector of the second rAAV particle is encapsidated by an Anc80 capsid.
  • the recombinant AAV vectors of the first and second rAAV particles are each independently encapsidated by an Anc80 capsid.
  • the Anc80 capsid comprises a polypeptide of SEQ ID NO: 109.
  • the present disclosure provides a composition
  • a composition comprising (a) a first rAAV particle comprising a recombinant AAV vector that comprises, in order of 5’ to 3’ : a 5’ ITR sequence of SEQ ID NO: 97; a CAG promoter comprising a CMV early enhancer element of SEQ ID NO: 98, a chicken beta actin gene sequence of SEQ ID NO: 99, and a chimeric intron of SEQ ID NO: 100; a 5OTOF coding region that comprises exons 1 to (and through) 21 of OTOF cDNA; a SD intron sequence of SEQ ID NO: 102; an AK recombinogenic sequence of SEQ ID NO: 103; and a 3’ ITR sequence of SEQ ID NO: 104; and (b) a second rAAV particle comprising a recombinant AAV vector that comprises a 5’ ITR sequence of SEQ ID NO: 97; an AK recombinogenic sequence of S
  • the 5’OTOF coding region is SEQ ID NO: 101. In some embodiments, the 5’OTOF coding region is at least 70% identical to SEQ ID NO: 101, and encodes the same amino acid sequence as SEQ ID NO: 101. In some embodiments, the 3 OTOF coding region is SEQ ID NO: 107.
  • the 3OTOF coding region is at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% identical to SEQ ID NO: 107, and encodes the same amino acid sequence as encoded by SEQ ID NO: 107.
  • the recombinant AAV vectors in the first and second rAAV particles are each independently encapsidated by an Anc80 capsid.
  • the Anc80 capsid comprises a polypeptide of SEQ ID NO: 109.
  • the recombinant AAV vectors of the first and second rAAV particles undergo concatamerization or homologous recombination with each other, thereby forming a recombined nucleic acid that encodes a full-length otoferlin protein within the cell.
  • the present disclosure provides a method comprising introducing into a cochlea of a mammal (e.g., a human) a therapeutically effective amount of any of the aforementioned composition.
  • a mammal e.g., a human
  • the mammal has been previously identified as having a defective otoferlin gene.
  • the present disclosure provides a method of increasing expression of a full-length otoferlin protein in a mammalian cell, the method comprising introducing any of the aforementioned compositions into the mammalian cell, e.g., an inner hair cell, e.g., a human cell.
  • the mammalian cell has previously been determined to have a defective otoferlin gene.
  • the present disclosure provides a method of increasing expression of a full-length otoferlin protein in an inner hair cell in a cochlea of a mammal, e.g., a human, the method comprising introducing into the cochlea of the mammal a therapeutically effective amount of any of the aforementioned compositions.
  • the mammal has been previously identified as having a defective otoferlin gene.
  • the present disclosure provides a method of treating non- symptomatic sensorineural hearing loss in a subject, e.g., a human identified as having a defective otoferlin gene, the method comprising administering a therapeutically effective amount of any of the aforementioned compositions into the cochlea of the subject.
  • the method further comprises, prior to the administering step, determining that the subject has a defective otoferlin gene.
  • the composition is administered to the cochlea using a microcatheter.
  • the microcatheter is shaped such that it can enter the middle ear cavity via the external auditory canal and contact the end of the microcatheter with the RWM.
  • the distal end of the microcatheter is comprised of at least one microneedle with diameter of between 10 and 1,000 microns.
  • the present disclosure provides a kit comprising any of the aforementioned compositions.
  • the composition is pre-loaded in a device, e.g., a microcatheter.
  • the microcatheter is shaped such that it can enter the middle ear cavity via the external auditory canal and contact the end of the microcatheter with the RWM.
  • the distal end of the microcatheter is comprised of at least one microneedle with diameter of between 10 and 1,000 microns.
  • DFNB9 a recessively inherited, non-syndromic prelingual hearing disorder.
  • Deficiency in otoferlin the protein encoded by OTOF , abolishes fast exocytosis from auditory inner hair cells (IHCs). Due the impairment of neurotransmission at the first auditory synapse, no sound signals are transmitted to the brain, explaining the profound deafness.
  • compositions and methods for treating non-symptomatic sensorineural hearing loss in a subject e.g., a human identified as having a defective otoferlin gene.
  • compositions that include at least two different nucleic acid vectors, where: each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, each of the encoded portions being at least 30 amino acid residues in length, where the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions; no single vector of the at least two different vectors encodes an active otoferlin protein (e.g., a full-length otoferlin protein); at least one of the coding sequences comprises a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons; and, when introduced into a mammalian cell, the at least two different vectors undergo homologous recombination with each other, thereby forming a recombined nucleic acid, where the recombined nucle
  • the recombined nucleic acid that encodes an active otoferlin protein exists as an episome in a mammalian cell (e.g., any of the types of mammalian cells described herein).
  • kits that include any of the compositions described herein.
  • methods that include introducing into a cochlea of a mammal a therapeutically effective amount of any of the compositions described herein.
  • an active otoferlin protein e.g., a full-length otoferlin protein
  • Also provided herein are methods of treating non- symptomatic sensorineural hearing loss in a subject identified as having a defective otoferlin gene that include: administering a therapeutically effective amount of any of the compositions described herein into the cochlea of the subject.
  • compositions, kits, and methods are described herein and can be used in any combination without limitation.
  • the human OTOF gene encodes otoferlin, which is a protein that, in some embodiments, plays a critical role in priming, fusion, and/or replenishing of synaptic vesicles of inner hair cell synapses during sound encoding.
  • otoferlin is a protein that, in some embodiments, plays a critical role in priming, fusion, and/or replenishing of synaptic vesicles of inner hair cell synapses during sound encoding.
  • otoferlin is a protein that, in some embodiments, plays a critical role in priming, fusion, and/or replenishing of synaptic vesicles of inner hair cell synapses during sound encoding.
  • Biallelic otoferlin gene mutations cause localized, synaptic transmission defects between hair cells and the auditory nerve. Otoferlin enables sensory cells to release neurotransmitters in response to stimulation by sound to activate auditory neurons and those neurons carry electronically encoded acousting information to the brain to produce “hearing.” When biallelic mutations in OTOF are present, that transmission is impaired and, as a result, a majority of subjects have congenital, severe to profound sensorineural hearing loss.
  • Otoacoustic emissions from DFNB9 subjects are normal, at least for the first decade of life, indicating morphological integrity of the inner ear and proper function of outer hair cells. Apart from the lack of synaptic transmission and the subsequent loss of synapses, the morphology and physiology of the inner ear remains preserved in DFNB9, at least during the first decade in life in humans. Accordingly, in some embodiments, restoration of OTOF and/or otoferlin function may serve to mitigate or prevent secondary degeneration of one or more cochlear structures.
  • mice with a random point mutation in the C2F domain short ( ⁇ 10ms) depolarizations of the IHCs elicited vesicle fusion of similar size as in wild type mice, however sustained stimulations uncovered a strong deficiency in replenishing vesicles to the readily releasable pool (Pangrsic et al. (2010) Nat. Neurosci. 13 869-876).
  • the p.Ile515Thr mutation found in human subjects with only mildly elevated hearing thresholds but a severe reduction in speech understanding and a temperature-dependent deafening (Varga et al.
  • Methods of detecting mutations in a gene are well-known in the art. Non-limiting examples of such techniques include: real-time polymerase chain reaction (RT-PCR), PCR, sequencing, Southern blotting, and Northern blotting.
  • RT-PCR real-time polymerase chain reaction
  • PCR PCR
  • sequencing Southern blotting
  • Northern blotting Northern blotting
  • the OTOF gene encodes otoferlin, a protein that is involved in synaptic vesicle exocytosis in cochlear hair cells (see, e.g., Johnson and Chapman (2010) J. Cell Biol. 191(1): 187-198; and Heidrych et al. (2008) Hum. Mol. Genet. 17:3814-3821).
  • the human OTOF gene is located on chromosome 2p23.3. It contains 48 exons encompassing ⁇ 132 kilobases (kb) (NCBI Accession No. NG009937.1).
  • the mRNA encoding the long-form of otoferlin expressed in the brain includes 48 exons (Yasunaga et ak, Am. J. Hum. Genet. 67:591-600, 2000). Forward and reverse primers that can be used to amplify each of the 48 exons in the OTOF gene are described in Table 2 of Yasunaga et ak, Am. J. Hum. Genet. 67:591-600, 2000.
  • the full- length OTOF protein is a full-length wildtype OTOF protein.
  • the full-length wildtype OTOF protein expressed from the human OTOF gene is 1997 residues in length.
  • An exemplary human wildtype otoferlin protein is or includes the sequence of any one of SEQ ID NOs: 1-5.
  • Isoform e of human otoferlin protein (SEQ ID NO: 5) is encoded by an mRNA that includes exon 48 and does not include exon 47 of the otoferlin gene (Yasunaga et ak, Am. J. Hum. Genet. 67:591-600, 2000).
  • the active otoferlin protein has the sequence of SEQ ID NO: 5, but is missing the 20 amino acids including the RXR motif identified in Strenzke et ak, EMBO J. 35(23):2499-2615, 2016.
  • Non-limiting examples of nucleic acids encoding a wildtype otoferlin protein are or include any one of SEQ ID NO: 7-11.
  • at least some or all of the codons in SEQ ID NO: 7-11 can be codon-optimized to allow for optimal expression in a non-human mammal or in a human.
  • Orthologs of human otoferlin proteins are known in the art.
  • otoferlin protein Human canonical (long) isoform sequence (otoferlin protein) (SEQ ID NO: 1) (also called otoferlin isoform a) (NCBI Accession No. AAD26117.1) Human Isoform 2 (short 1) (otoferlin protein) (SEQ ID NO: 2) (also called otoferlin isoform d) (NCBI Accession No. NP_919304.1)
  • Human Isoform 3 (short 2) (otoferlin protein) (SEQ ID NO: 3) (also called otoferlin isofom c) (NCBI Accession No. NP_919303.1)
  • Human Isoform 4 (short 3) (otoferlin protein) (SEQ ID NO: 4) (also called otoferlin isoform b) (NCBI Accession No. NP_004793.2)
  • Human Isoform 5 (short 4) (otoferlin protein) (SEQ ID NO: 5) (also called otoferlin isoform e) (NCBI Accession No. NP_001274418.1)
  • SEQ ID NO: 12 A non-limiting example of a human wildtype otoferlin genomic DNA sequence is SEQ ID NO: 12.
  • the exons in SEQ ID NO: 12 are: nucleotide positions 5001-5206 (exon 1), nucleotide positions 25925-25983 (exon 2), nucleotide positions 35779-35867 (exon 3), nucleotide positions 44590-44689 (exon 4), nucleotide positions 47100-47281 (exon 5), nucleotide positions 59854-59927 (exon 6), nucleotide positions 61273-61399 (exon 7), nucleotide positions 61891-61945 (exon 8), nucleotide positions 68626- 68757(exon 9), nucleotide positions 73959-74021 (exon 10), nucleotide positions 74404- 74488 (exon 11), nucleotide positions 79066-79225 (exon 12), nucleotide positions
  • the introns are located between each contiguous pair of exons in SEQ ID NO: 12, i.e., at nucleotide positions 100-5001 (intron 1), nucleotide 5207-25924 (intron 2), nucleotide positions 25984-35778 (intron 3), nucleotide positions 35868-44589 (intron 4), nucleotide positions 44690-47099 (intron 5), nucleotide positions 47282-59853(intron 6), nucleotide positions 59928-61272 (intron 7), nucleotide positions 61400-61890 (intron 8), nucleotide positions 61946-68625 (intron 9), nucleotide positions 68758- 73958 (intron 10), nucleotide positions 74022-74403 (intron 11), nucleotide positions 74489-79065 (intron 12), nucleotide positions 79226-80050 (intron 13), nucleotide positions 802
  • an otoferlin gene may be split into two or more segments between or within any appropriate exons and/or introns, where each segment is included in a different vector of the present disclosure.
  • the otoferlin gene is split at exon 21, i.e., with exons 1 to (and through) 21 in a first vector and exons 22 to (and through) exon 48 in a second vector.
  • the otoferlin segments in the first and second vectors are derived from an otoferlin cDNA sequence and lack introns, i.e., with exons 1 to (and through) 21 in a first vector and exons 22 to (and through) exon 48 in a second vector, each vector lacking otoferlin introns.
  • an otoferlin gene may be split at one or more other exons and/or introns as long as, when combined with all other components of a vector, the packaging capacity of the vector is not exceeded.
  • Rat Otoferlin Protein SEQ ID NO: 20
  • Cow Otoferlin Protein (SEQ ID NO: 22)
  • a first vector comprises a 5’ portion of OTOF cDNA, e.g., as shown in SEQ ID NO: 94.
  • a second vector comprises a 3’ portion of OTOF cDNA, e.g., as shown in SEQ ID NO: 95.
  • compositions described herein can include a first vector including the coding sequence of SEQ ID NO: 94 (or include a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 94).
  • compositions described herein can include a second vector including the coding sequence of SEQ ID NO: 95 (or include a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 95).
  • compositions described herein can include a first vector with a 5’ OTOF coding region that comprises exons 1 to (and through) 21 of OTOF cDNA.
  • first vector that comprises the nucleotide sequence of SEQ ID NO: 101 (or a sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 101).
  • compositions described herein can include a first vector that comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 101, and encodes the same amino acid sequence as SEQ ID NO: 101.
  • a composition of the present disclosure includes a first vector that comprises a codon optimized version of SEQ ID NO: 101, i.e., a nucleotide sequence that encodes the same amino acid sequence as SEQ ID NO: 101 but with codons that have been optimized for expression in a particular cell type, e.g., a mammalian cell, e.g., a human cell.
  • the first vector does not include any other portion of an OTOF gene.
  • the first vector does not include any other portion of OTOF cDNA.
  • compositions described herein can include a second vector with a 3’ OTOF coding region that comprises exons 22 to (and through) exon 48 of OTOF cDNA.
  • compositions described herein can include a second vector that comprises the nucleotide sequence of SEQ ID NO: 108 (or a sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 108).
  • compositions described herein can include a second vector that comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 108, and encodes the same amino acid sequence as SEQ ID NO: 108.
  • a composition of the present disclosure includes a second vector that comprises a codon optimized version of SEQ ID NO: 108, i.e., a nucleotide sequence that encodes the same amino acid sequence as SEQ ID NO: 108 but with codons that have been optimized for expression in a particular cell type, e.g., a mammalian cell, e.g., a human cell.
  • the second vector does not include any other portion of an OTOF gene.
  • the second vector does not include any other portion of OTOF cDNA.
  • nucleic acid therapeutics such as auditory polypeptide messenger RNAs.
  • the auditory polypeptide nucleic acids are present in viral vectors, such as adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, and retroviral vectors.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • efficient RNA processing signals such as splicing and polyadenylation (poly A) signals
  • sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • a great number of expression control sequences including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
  • nucleic acid sequence e.g., coding sequence
  • regulatory sequences are said to be “operably” linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences.
  • nucleic acid sequences be translated into a functional protein
  • two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • operably linked coding sequences yield a fusion protein.
  • operably linked coding sequences yield a functional RNA (e.g., shRNA, miRNA, miRNA inhibitor).
  • a polyadenylation sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence.
  • a rAAV construct useful in the present disclosure may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene.
  • One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence.
  • One possible intron sequence has the nucleotide sequence of SEQ ID NO: 100.
  • Another vector element that may be used is an internal ribosome entry site (IRES).
  • An IRES sequence is used to produce more than one polypeptide from a single gene transcript.
  • An IRES sequence would be used to produce a protein that contains more than one polypeptide chain.
  • a Foot and Mouth Disease Virus 2A sequence is included in polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p.
  • the precise nature of the regulatory sequences needed for gene expression in host cells may vary between species, tissues or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the disclosure may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., RSV LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., cytomegalovirus
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art.
  • inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al. (1996) Proc. Natl. Acad. Sci. USA, 93:3346-3351), the tetracycline- repressible system (Gossen et al. Proc. Natl. Acad. Sci.
  • MT zinc-inducible sheep metallothionine
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system WO 98/10088
  • ecdysone insect promoter No et al. (1996) Proc. Natl. Acad. Sci. USA, 93:3346-3351
  • inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • the native promoter for the transgene will be used.
  • the native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression.
  • the native promoter may be used when expression of the transgene must be regulated temporally or developmental ⁇ , or in a tissue-specific manner, or in response to specific transcriptional stimuli.
  • other native expression control elements such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.
  • the regulatory sequences impart tissue-specific gene expression capabilities.
  • the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner.
  • tissue-specific regulatory sequences e.g., promoters, enhancers, etc.
  • tissue-specific regulatory sequences are well known in the art.
  • tissue-specific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter (Kugler et al., Virology 311:89-95, 2003; Hioki et al., Gene Ther. 14:872-882, 2007; Kuroda et al., ./. Gene Med.
  • TBG liver-specific thyroxin binding globulin
  • PPY pancreatic polypeptide
  • Syn synapsin-1
  • a creatine kinase (MCK) promoter Wang et al., Gene Ther. 15:1489-1499, 2008; Talbot et al., Mol. Ther. 18:601-608, 2010; Katwal et al., Gene Ther. 20(9):930-938, 2013
  • DES mammalian desmin
  • a C5-12 promoter Wang et al., Gene Ther. 15:1489- 1499, 2008
  • a-MHC a-myosin heavy chain promoter
  • PDGF promoter Pursumna, Gene Ther.
  • MecP2 promoter Rastegar et al., PLoS One 4:e6810, 2009; Gray et al., Human Gene Ther. 22:1143-1153, 2011
  • CaMKII promoter Hioki et al., Gene Ther. 14:872-882, 2007; Kuroda et al., J. Gene Med. 10:1163-1175, 2008
  • mGluR2 promoter Brene et al., Eur. J. Eurosci. 12:1525-1533, 2000; Kuroda et al., J. Gene Med.
  • NFL promoter Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332, 2001
  • NFH promoter Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332, 2001
  • nJ2 promoter Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332, 2001
  • PPE promoter Xu et al., Human Gene Ther.
  • exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum.
  • bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor .alpha. -chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiok, 13:503-15 (1993); Xu et al., Human Gene Ther.
  • one or more bindings sites for one or more miRNAs are incorporated in a transgene of a rAAV vector, to inhibit the expression of the transgene in one or more tissues of an subject harboring the transgene.
  • binding sites may be selected to control the expression of a transgene in a tissue specific manner.
  • binding sites for the liver-specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver.
  • the target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region. Typically, the target site is in the 3' UTR of the mRNA.
  • the transgene may be designed such that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites.
  • the presence of multiple miRNA binding sites may result in the cooperative action of multiple RISCs and provide highly efficient inhibition of expression.
  • the target site sequence may comprise a total of 5-100, 10-60, or more nucleotides.
  • the target site sequence may comprise at least 5 nucleotides of the sequence of a target gene binding site.
  • composition of the auditory polypeptide transgene sequence of the rAAV vector will depend upon the use to which the resulting vector will be generated.
  • the disclosure embraces the delivery of rAAV vectors encoding one or more auditory polypeptides, peptides, or proteins, which are useful for the treatment or prevention of disease states associated with hearing loss in a mammalian subject.
  • Exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of otoferlin, Ca v 1.3, a scaffold protein selected from bassoon, piccolo, ribeye, and harmonin, Vglut3, synaptotagmin, a vesicle tethering / docking protein, a vesicle priming protein, a vesicle fusion protein, GluA2/3, or GluA4.
  • Reporter sequences that may be provided in a transgene include, without limitation, DNA sequences encoding .beta. -lactamase, .beta.-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • the reporter sequences When associated with regulatory elements which drive their expression, the reporter sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays, and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunohistochemistry for example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for J- galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.
  • Such reporters can, for example, be useful in verifying the tissue-specific targeting capabilities and tissue specific promoter regulatory activity of an rAAV.
  • the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of disease.
  • Appropriate transgene coding sequences will be apparent to the skilled artisan.
  • the rAAV vectors may comprise a gene or a portion of a gene encoding an auditory polypeptide, to be transferred to a subject to treat a disease associated with reduced expression, lack of expression or dysfunction of the auditory polypeptide gene or another gene in the functional pathway of an auditory polypeptide.
  • therapeutic compositions including a plurality of adeno-associated viral (AAV) vectors, wherein the plurality of AAV vectors are capable of constituting an auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • the plurality of AAV vectors are capable of constituting a full-length auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • the plurality of AAV vectors include a first AAV vector and a second AAV vector, wherein the first and second AAV vectors independently contain packaging capacity of less than about 6kb.
  • the auditory polypeptide messenger RNA encodes an auditory polypeptide selected from the group consisting of otoferlin and an ortholog or homolog thereof, for example, as provided herein.
  • the AAV vectors contain at least one promoter sequence selected from a CBA, a CMV, or a CB7 promoter, or one or more Cochlea-specific promoters.
  • an AAV vector can include a CMV enhancer and promoter sequence, e.g., SEQ ID NO: 70.
  • an AAV vector can include a CMV enhancer and a chicken b-actin (CBA) promoter, e.g., SEQ ID NO: 61.
  • CBA chicken b-actin
  • an AAV vector can include a CMVd promoter sequence, e.g., SEQ ID NO: 86.
  • an AAV vector can include a promoter that comprises a CMV enhancer and a CBA promoter, e.g., the nucleotide sequences of SEQ ID NO: 98 and 99.
  • the nucleotide sequence of SEQ ID NO: 98 precedes the nucleotide sequence of SEQ ID NO: 99 and is optionally followed by a chimeric intron, e.g., the nucleotide sequence of SEQ ID NO: 100.
  • compositions provided herein include at least two (e.g., two, three, four, five, or six) nucleic acid vectors, where: each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, each of the encoded portions being at least 30 amino acids (e.g., between about 30 amino acids to about 1950 amino acids, about 30 amino acids to about 1900 amino acids, about 30 amino acids to about 1850 amino acids, about 30 amino acids to about 1800 amino acids, about 30 amino acids to about 1750 amino acids, about 30 amino acids to about 1700 amino acids, about 30 amino acids to about 1650 amino acids, about 30 amino acids to about 1600 amino acids, about 30 amino acids to about 1550 amino acids, about 30 amino acids to about 1500 amino acids, about 30 amino acids to about 1450 amino acids, about 30 amino acids to about 1400 amino acids, about 30 amino acids to about 1350 amino acids, about 30 amino acids to about 1300 amino acids, about 30 amino acids to about 1250 amino acids, about 30 amino acids to about 1200
  • one of the nucleic acid vectors can include a coding sequence that encodes a portion of an otoferlin protein, where the encoded portion is, e.g., about 900 amino acids to about 1950 amino acids, about 900 amino acids to about 1900 amino acids, about 900 amino acids to about 1850 amino acids, about 900 amino acids to about 1800 amino acids, about 900 amino acids to about 1750 amino acids, about 900 amino acids to about 1700 amino acids, about 900 amino acids to about 1650 amino acids, about 900 amino acids to about 1600 amino acids, about 900 amino acids to about 1550 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1450 amino acids, about 900 amino acids to about 1400 amino acids, about 900 amino acids to about 1350 amino acids, about 900 amino acids to about 1300 amino acids, about 900 amino acids to about 1250 amino acids, about 900 amino acids to about 1200 amino acids, about 900 amino acids to about 1150 amino acids, about 900 amino acids to
  • At least one of the coding sequences includes a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks the intronic sequence that naturally occurs between the two neighboring exons.
  • the amino acid sequence of none of the encoded portions overlaps even in part with the amino acid sequence of a different one of the encoded portions. In some embodiments, the amino acid sequence of one or more of the encoded portions partially overlaps with the amino acid sequence of a different one of the encoded portions. In some embodiments, the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different one of the encoded portions.
  • the overlapping amino acid sequence is between about 30 amino acid residues to about 1000 amino acids (e.g., or any of the subranges of this range described herein) in length.
  • the vectors include two different vectors, each of which comprises not only exon(s), but also a different segment of an intron, wherein the intron includes the nucleotide sequence of an intron that is present in an otoferlin genomic DNA (e.g., any of the exemplary introns in SEQ ID NO: 12 described herein), and wherein the two different segments overlap in sequence by at least 100 nucleotides (e.g., about 100 nucleotides to about 5,000 nucleotides, about 100 nucleotides to about 4,500 nucleotides, about 100 nucleotides to about 4,000 nucleotides, about 100 nucleotides to about 3,500 nucleotides, about 100 nucleotides to about 3,000 nucleotides, about 100 nucleotides to about 2,500 nucleotides, about 100 nucleotides to about 2,000 nucleotides, about 100 nucleotides to about 1,500 nucleotides, about 100 nucleotides
  • nucleotides to about 3,500 nucleotides about 1,500 nucleotides to about 3,000 nucleotides, about 1,500 nucleotides to about 2,500 nucleotides, about 1,500 nucleotides to about 2,000 nucleotides, about 2,000 nucleotides to about 5,000 nucleotides, about 2,000 nucleotides to about 4,500 nucleotides, about 2,000 nucleotides to about 4,000 nucleotides, about 2,000 nucleotides to about 3,500 nucleotides, about 2,000 nucleotides to about 3,000 nucleotides, about 2,000 nucleotides to about 2,500 nucleotides, about
  • nucleotides to about 5,000 nucleotides about 2,500 nucleotides to about 4,500 nucleotides, about 2,500 nucleotides to about 4,000 nucleotides, about 2,500 nucleotides to about 3,500 nucleotides, about 2,500 nucleotides to about 3,000 nucleotides, about 3,000 nucleotides to about 5,000 nucleotides, about 3,000 nucleotides to about 4,500 nucleotides, about 3,000 nucleotides to about 4,000 nucleotides, about 3,000 nucleotides to about 3,500 nucleotides, about 3,500 nucleotides to about 5,000 nucleotides, about 3,500 nucleotides to about 4,500 nucleotides, about 3,500 nucleotides to about 4,000 nucleotides, about 4,000 nucleotides to about 5,000 nucleotides, about 4,000 nucleotides to about 4,500 nucleotides, about 4,500 nucleo
  • the overlapping nucleotide sequence in any two of the different vectors can include part or all of one or more exons of an otoferlin gene (e.g., any one or more of the exemplary exons in SEQ ID NO: 12 described herein).
  • the number of different vectors in the composition is two, three, four, or five.
  • the first of the two different vectors can include a coding sequence that encodes an N-terminal portion of the otoferlin protein.
  • the N- terminal portion of the otoferlin gene is between about 30 amino acids to about 1950 amino acids (or any of the subranges of this range described above) in length.
  • the first vector further includes one or both of a promoter (e.g., any of the promoters described herein or known in the art) and a Kozak sequence (e.g., any of the exemplary Kozak sequences described herein or known in the art).
  • the first vector includes a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.
  • the second of the two different vectors includes a coding sequence that encodes a C-terminal portion of the otoferlin protein.
  • the C-terminal portion of the otoferlin protein is between 30 amino acids to about 1950 amino acids (or any of the subranges of this range described above) in length.
  • the second vector further includes a poly(A) signal sequence.
  • the N-terminal portion encoded by one of the two vectors can include a portion comprising amino acid position 1 to about amino acid position 1,950, about amino acid position 1,940, about amino acid position 1,930, about amino acid position 1,920, about amino acid position 1,910, about amino acid position 1,900, about amino acid position 1,900, about amino acid position 1,890, about amino acid position 1,880, about amino acid position 1,870, about amino acid position 1,860, about amino acid position 1,850, about amino acid position 1,840, about amino acid position 1,830, about amino acid position 1,820, about amino acid position 1,810, about amino acid position 1,800, about amino acid position 1,790, about amino acid position 1,780, about amino acid position 1,770, about amino acid position 1,760, about amino acid position 1,750, about amino acid position 1,740, about amino acid position 1,730, about amino acid position 1,720, about amino acid position 1,710, about amino acid position 1,700, about amino acid position, about amino acid position, about amino acid
  • the N-terminal portion of the precursor otoferlin protein can include a portion comprising amino acid position 1 to amino acid position 310, amino acid position 1 to about amino acid position 320, amino acid position 1 to about amino acid position 330, amino acid position 1 to about amino acid position 340, amino acid position 1 to about amino acid position 350, amino acid position 1 to about amino acid position 360, amino acid position 1 to about amino acid position 370, amino acid position 1 to about amino acid position 380, amino acid position 1 to about amino acid position 390, amino acid position 1 to about amino acid position 400, amino acid position 1 to about amino acid position 410, amino acid position 1 to about amino acid position 420, amino acid position 1 to about amino acid position 430, amino acid position 1 to about amino acid position 440, amino acid position 1 to about amino acid position 450, amino acid position 1 to about amino acid position 460, amino acid position 1 to about amino acid position 470, amino acid position 1 to about amino acid position 480, amino acid position 1
  • the composition includes two vectors, where a first of the two vectors includes a coding sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 62, and the second of the two vectors includes a coding sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 63.
  • vector includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that is capable of replication when associated with the proper control elements and that can transfer gene sequences between cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter.
  • a vector can be an artificial chromosome (e.g., a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a Pl-derived artificial chromosome (PAC)) or a viral vector (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), any retroviral vectors as described herein, and any Gateway® vectors).
  • a vector can, e.g., include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence (e.g., a gene).
  • the phrases “operatively positioned,” “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid coding sequence is capable of being transcribed.
  • expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitor) from a transcribed gene.
  • Vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the nucleic acids described herein.
  • the vector is a plasmid (i.e. a circular DNA molecule that can autonomously replicate inside a cell).
  • the vector can be a cosmid (e.g., pWE and sCos series (Wahl et al. (1987) Proc. Natl. Acad. Sci. USA 84:2160-2164, Evans et al. (1989) Proc. Natl. Acad. Sci. USA 86:5030-5034).
  • transfer vector refers to a composition of matter which includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “transfer vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector including a recombinant polynucleotide including expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • the vector(s) is an artificial chromosome.
  • An artificial chromosome is a genetically engineered chromosome that can be used as a vector to carry large DNA inserts.
  • the artificial chromosome is human artificial chromosome (HAC) (see, e.g., Kouprina et al., Expert Opin. DrugDeliv 11(4): 517-535, 2014; Basu et al., Pediatr. Clin. North Am. 53: 843-853, 2006; Ren et al., Stem. Cell Rev. 2(l):43-50, 2006; Kazuki et al., Mol. Ther.
  • HAC human artificial chromosome
  • the vector(s) is a yeast artificial chromosome (YAC) (see, e.g., Murray et al., Nature 305: 189-193, 1983; Ikeno et al. (1998) Nat. Biotech. 16:431- 439, 1998).
  • the vector(s) is a bacterial artificial chromosome (BAC) (e.g., pBeloBACl 1, pECBACl, and pBAC108L).
  • BAC bacterial artificial chromosome
  • the vector(s) is a Pl-derived artificial chromosome (PAC). Examples of artificial chromosome are known in the art.
  • the vector(s) is a viral vector (e.g., adeno-associated virus, adenovirus, lentivirus, and retrovirus).
  • a viral vector e.g., adeno-associated virus, adenovirus, lentivirus, and retrovirus.
  • Non-limiting examples of viral vectors are described herein.
  • “Recombinant AAV vectors” or “rAAVs” of the disclosure are typically comprised of, at a minimum, a transgene or a portion thereof and a regulatory sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell.
  • the transgene is a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
  • the nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a cell of a target tissue.
  • the vector(s) is an adeno-associated viral vector (AAV) (see, e.g., Asokan et ah, Mo/. Ther. 20: 699-7080, 2012).
  • AAV adeno-associated viral vector
  • “Recombinant AAV vectors” or “rAAVs” are typically composed of, at a minimum, a transgene or a portion thereof and a regulatory sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs).
  • ITRs optionally 5' and 3' AAV inverted terminal repeats
  • Such a recombinant AAV vector is packaged into a capsid to form an rAAV particle and delivered to a selected target cell (e.g., an inner hair cell).
  • the AAV sequences of the vector typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)).
  • the ITR sequences are about 145 nt in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al. "Molecular Cloning.
  • an ITR is or comprises 145 nucleotides.
  • an ITR is a wild-type AAV2 ITR, e.g., the 5’ ITR of SEQ ID NO: 97 and the 3’ ITR of SEQ ID NO: 104.
  • an ITR is derived from a wild-type AAV2 ITR and includes one or more modifications, e.g., truncations, deletions, substitutions or insertions as is known in the art.
  • an ITR comprises fewer than 145 nucleotides, e.g., 127, 130, 134 or 141 nucleotides.
  • an ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 ,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 144, or 145 nucleotides.
  • a non-limiting example of a 5’ AAV ITR sequence is SEQ ID NO: 59.
  • a non limiting example of a 3’ AAV ITR sequence is SEQ ID NO: 60.
  • vectors and/or constructs of the present disclosure comprise a 5’ AAV ITR and/or a 3’ AAV ITR.
  • a 5’ AAV ITR sequence is SEQ ID NO: 97.
  • a 3’ AAV ITR sequence is SEQ ID NO: 104.
  • the 5’ AAV ITR sequence is SEQ ID NO: 97 and the 3’ AAV ITR sequence is SEQ ID NO: 104.
  • the 5’ and a 3’ AAV ITRs flank a portion of a transgene and/or construct comprising a portion of OTOF (e.g., SEQ ID NO: 101 or 107).
  • the vector also includes conventional control elements that are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the disclosure.
  • operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • AAV vectors as described herein may include any of the regulatory elements described herein (e.g., one or more of a promoter, a polyA sequence, and an IRES).
  • one or more recombinant AAV vectors of the present disclosure is packaged into a capsid of the AAV2, 3, 4, 5, 6, 7, 8, 9, 10, rh8, rhlO, rh39, rh43 or Anc80 serotype or one or more hybrids thereof.
  • a capsid is from an ancestral serotype.
  • the capsid is an Anc80 capsid (e.g., an Anc80L65 capsid).
  • the capsid comprises a polypeptide represented by SEQ ID NO: 109.
  • the capsid comprises a polypeptide with at least 85%, 90%, 95%, 98% or 99% sequence identity to the polypeptide represented by SEQ ID NO: 109.
  • ITRs and capsids may be used in recombinant AAV vectors of the present disclosure, for example, wild-type or variant AAV2 ITRs and Anc80 capsid, wild-type or variant AAV2 ITRs and AAV6 capsid, etc.
  • an rAAV particle is an rAAV2/Anc80 particle which comprises an Anc80 capsid (e.g., comprising a polypeptide of SEQ ID NO: 109) that encapsidates a nucleic acid vector with wild-type AAV2 ITRs (e.g., SEQ ID NOs: 97 and 104) flanking a portion of a transgene and/or construct comprising a portion of OTOF (e.g., SEQ ID NO: 101 or 107).
  • Anc80 capsid e.g., comprising a polypeptide of SEQ ID NO: 109
  • wild-type AAV2 ITRs e.g., SEQ ID NOs: 97 and 104
  • compositions including one or more adenoviral (AV) vectors, wherein the one or the plurality of AV vectors are capable of constituting an auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • the one or the plurality of AV vectors are capable of constituting a full-length auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • the vector(s) is an adenovirus (see, e.g., Dmitriev et al. (1998) J. Virol. 72: 9706-9713; and Poulin et al., J. Virol % : 10074-10086, 2010).
  • the vector(s) is a retrovirus (see, e.g., Maier et al. (2010) Future Microbiol 5: 1507-23).
  • the vector(s) is a lentivirus (see, e.g., Matrai et al. (2010) Mol Ther. 18: 477-490; Banasik et al. (2010) Gene Ther. 17:150-7; and Wanisch et al. (2009) Mol. Ther. 17: 1316-32).
  • a lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Non-limiting lentivirus vectors that may be used in the clinic include the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen, and the like. Other types of lentiviral vectors are also available and would be known to one skilled in the art.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, e.g., the LENTIVECTOR®. gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • compositions including one or a plurality of lentiviral vectors, wherein the one or the plurality of lentiviral vectors are capable of constituting an auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • the one or the plurality of lentiviral vectors are capable of constituting a full-length auditory polypeptide messenger RNA in a target cell of a human subject to whom the therapeutic composition is administered.
  • the first vector includes an ITR (e.g., any of the exemplary ITR sequences described herein), a promoter and/or enhancer (e.g., any of the exemplary enhancers and any of the exemplary promoters described herein), an intron sequence of a OTOF gene (e.g., a human OTOF gene, e.g., any of the exemplary intron sequences of a human OTOF gene described herein), a Kozak sequence (e.g., any of the exemplary Kozak sequences described herein), and a sequence encoding a first, N-terminal portion of a human otoferlin protein (e.g., any of the exemplary sequences encoding a first, N-terminal portion of a human otoferlin protein described herein), an AK sequence (e.g., any of the exemplary AK sequences described herein), and an ITR (e.g., any of the exemplary ITR (e.g., any
  • the second vector includes an ITR sequence (e.g., any of the exemplary ITR sequences described herein), an AK sequence (e.g., any of the exemplary AK sequences described herein), a splicing acceptor sequence (e.g., any of the splicing acceptor sequences described herein), a sequence encoding a second portion of a human otoferlin protein (e.g., any of the exemplary sequences encoding a second, C-terminal portion of a human otoferlin protein described herein), a poly(A) signal sequence (e.g., any of the exemplary poly(A) signal sequences described herein), a stuff er sequence (e.g., any of the exemplary stuffer sequences described herein), and an ITR sequence (e.g., any of the exemplary ITR sequences described herein).
  • an ITR sequence e.g., any of the exemplary ITR sequences described herein
  • an AK sequence e.g
  • the vector is pAAV-AK-SA-3’mOTOF-EWB (SEQ ID NO: 39), depicted in Figures 11, 17 and 56, or is a vector including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 39.
  • the pAAV-AK-SA-3’mOTOF-EWB vector is 7625 bp in length and has an AK sequence at nucleotide positions 2-78, a splicing acceptor (SA) site at nucleotide positions 79-129, a 3’ mOTOF at nucleotide positions 130-3540, C2D at nucleotide positions 490-891, C2E at nucleotide positions 1996-2516, C2F at nucleotide positions 2749-3234, a WPRE at nucleotide positions 3595-4188, an ampicillin (AMP) resistance gene at nucleotide positions 5537-6537, a bovine growth hormone poly A-tail (bGH pA) at nucleotide positions 4212-4422, a phage-derived fl(+) origin of replication (ORI) at nucleotide positions 4674-5133, an origin of replication (ORI) at nucleotide positions 6787-7012.
  • the vector is pAAV-SA-3’mOTOF-EWB (SEQ ID NO: 40), depicted in Figures 12, 20 and 54, or is a vector including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 40.
  • the pAAV-SA-3’mOTOF-EWB vector is 7548 bp in length and has a splicing acceptor (SA) site at nucleotide positions 2-52, a 3’ mOTOF at nucleotide positions 53-3463, C2D at nucleotide positions 413-814, C2E at nucleotide positions 1919-2439, C2F at nucleotide positions 2672-3157, a WPRE at nucleotide positions 3518-4111, an ampicillin (AMP) resistance gene at nucleotide positions 5460-6460, a bovine growth hormone poly A-tail (bGH pA) at nucleotide positions 4135-4345, and a phage-derived fl(+) origin of replication (ORI) at nucleotide positions 4597-5056.
  • SA splicing acceptor
  • the vector is p AAV -HB A-eGFP-P2 A-5 ’ mOTOF - SD (SEQ ID NO: 41), depicted in Figures 13, 18 and 53, or is a vector including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 41.
  • the pAAV-HBA-eGFP-P2A- 5’mOTOF-SD vector is 7346 bp in length and has a Kozak sequence(*) at nucleotide positions 662-667, an enhanced green fluorescent protein (eGFP) sequence at nucleotide positions 668-1384, a P2A at nucleotide positions 1391-1456, a Kozak sequence at nucleotide positions 1463-1468, a 5’ mOTOF sequence at nucleotide positions 1469- 3988, a C2A at nucleotide positions 1469-1831, a C2B at nucleotide positions 2231- 2599, a C2C at nucleotide positions 2720-3091, human OTOF exon 21 at nucleotide positions 3872-3988, a splicing donor (SD) site at nucleotide positions 3989-4070, an AMP resistance gene at nucleotide positions 5186-6186, a fl
  • 19 and 55 is a vector including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 42.
  • the pAAV-HBA-eGFP- P2A-5’mOTOF-SD-AK is 7423 bp in length and has a Kozak sequence(*) at nucleotide positions 662-667, an enhanced green fluorescent protein (eGFP) sequence at nucleotide positions 668-1384, a P2A at nucleotide positions 1391-1456, a Kozak sequence at nucleotide positions 1463-1468, a 5’ mOTOF sequence at nucleotide positions 1469- 3988, a C2A at nucleotide positions 1469-1831, a C2B at nucleotide positions 2231- 2599, a C2C at nucleotide positions 2720-3091, human OTOF exon 21 at nucleotide positions 3872-3988, a splicing donor (SD) site at nucleotide positions 3989-4070, an AK sequence at nucleotide positions 4071-4147, an AMP resistance gene at
  • the vector is pAKOS102 (SEQ ID NO: 43), or is a vector including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 43.
  • pAKOS102 is shown in Figure 21.
  • the pAKOS102 vector is 7006 bp in length and has an AAV ITR sequence at nucleotide positions 1-130, a factor VIII stuffer sequence at nucleotide positions 145-1144, a human cytomegalovirus (hCMV) enhancer at nucleotide positions 1145-1486, a human ACTB promoter at nucleotide positions 1487-1869, a Kozak sequence at nucleotide positions 1883-1888, a 5’ hOTOF isoform 5 sequence at nucleotide positions 1889-4411, a SD intron sequence at nucleotide positions 4412-4493, an AAV2 ITR sequence at nucleotide positions 4519-4659, a fl(+)ORI at nucleotide positions 4734-5189, an KAN resistance gene at nucleotide positions 5469-6278, and an ORI at nucleotide positions 6357-6945.
  • AAV ITR sequence at nucleotide positions
  • the vector is pAKOSKM (SEQ ID NO: 45), or is a vector including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 45.
  • pAKOS104 is shown in Figure 24.
  • the pAKOS104 vector is 7083 bp in length and has an AAV ITR sequence at nucleotide positions 1-130, a factor VIII stuffer sequence at nucleotide positions 145-1144, a human cytomegalovirus (hCMV) enhancer at nucleotide positions 1145-1486, a human ACTB promoter at nucleotide positions 1487-1869, a Kozak sequence at nucleotide positions 1883-1888, a 5’ hOTOF isoform 5 sequence at nucleotide positions 1889-4411, a SD intron sequence at nucleotide positions 4412-4493, an AK sequence at nucleotide positions 4494-4570, an AAV2 ITR sequence at nucleotide positions 4596-4736, a fl(+)ORI at nucleotide positions 4811-5266, an KAN resistance gene at nucleotide positions 5546-6355, and an ORI at nucleotide positions 6434-70
  • the vector is pAKOS105_GFP (SEQ ID NO: 48), depicted in Figure 29, or is a vector including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 48.
  • the pAKOS105_GFP vector is 7761 bp in length and has an AAV ITR sequence at nucleotide positions 1-130, an AK sequence at nucleotide positions 145-221, a SA site sequence at nucleotide positions 222-272, a 3’ hOTOF isoform 5 sequence at nucleotide positions 273-3740, a T2A sequence at nucleotide positions 3750-3803, a turboGFP sequence at nucleotide positions 3804-4499, a bGH poly(A) signal at nucleotide positions 4509-4748, a factor VIII stuffer sequence at nucleotide positions 4749-5248, an AAV2 ITR sequence at nucleotide positions 5274- 5414, a fl(+)ORI at nucleotide positions 5489-5944, an KAN resistance gene at nucleotide positions 6224-7033, and an ORI at nucleotide positions 7112-7700.
  • the vector is pAKOS109 (SEQ ID NO: 52), or is a vector including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52.
  • the pAKOS109 vector is 7056 bp in length and has an AAV ITR sequence at nucleotide positions 1-130, a human cytomegalovirus (hCMV) enhancer at nucleotide positions 145-524, a chicken b-actin promoter at nucleotide positions 527-802, a chimeric intron at nucleotide positions 803-1815, a Kozak sequence at nucleotide positions 1856-1861, a 5’ hOTOF isoform 5 sequence at nucleotide positions 1862-4384, a SD intron sequence at nucleotide positions 4385-4466, an AK sequence at nucleotide positions 4467-4543, an AAV2 ITR sequence at nucleotide positions 4569-4709, a fl(+)ORI at nucleotide positions 4784-5239, an KAN resistance gene at nucleotide positions 5519-6328, and an ORI
  • the vector is pl09 (SEQ ID NO: 84) or is a vector that includes a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 84.
  • the pl09 vector is 4,711 bp in length and is shown in Figure 38.
  • the vector is pl05 (SEQ ID NO: 85) or is a vector that includes a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 85.
  • the pl05 vector is 4,664 bp in length and is shown in Figures 28 and 39.
  • the vector is 105.WPRE, shown in Figure 40.
  • the WPRE sequence present in the 105.WPRE vector is SEQ ID NO: 69.
  • the vector is pi 08, shown in Figure 41.
  • the pi 08 vector includes the FVIII stuffer sequence of SEQ ID NO: 58 and the CMV enhancer and promoter sequence of SEQ ID NO: 70.
  • the vector is 10TOF18.CL1, shown in Figure 42.
  • the 10TOF18.CL1 vector includes the CL1 degradation sequence of SEQ ID NO: 71.
  • the vector is 190T0F48, shown in Figure 43.
  • the vector is 1OTOF20.CL1, shown in Figure 44.
  • the intron 21 splice donor sequence in lOTOF20,CLl is SEQ ID NO: 72.
  • the vector is 210T0F48.WPRE, shown in Figure 45.
  • the intron 21 splice acceptor sequence is SEQ ID NO: 73.
  • the vector is 10T0F21.CL1, shown in Figure 46.
  • the intron 22 splice donor sequence is SEQ ID NO: 74.
  • the vector is 220T0F48.WPRE, shown in Figure 47.
  • the intron 22 splice acceptor sequence is SEQ ID NO: 75.
  • the vector is 105.pA.NTF3.CMVd, shown in Figure 48.
  • the 105.pA.NTF3.CMVd vector includes the following sequences: SV40 poly A (SEQ ID NO: 76), HSV-TK poly (A) (SEQ ID NO:
  • sequence encoding human NTF3 (SEQ ID NO: 79), and CMVd (SEQ ID NO: 86).
  • a vector can include a CMV enhancer and a chicken J- actin promoter, e.g., a sequence of SEQ ID NO: 61.
  • the vector is pAAV-HBA-eGFP-P2A-5’mOTOF.SD (SEQ ID NO: 87), or is a vector that includes a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 87.
  • the pAAV-HBA-eGFP-P2A-5’mOTOF.SD vector is 4,472 bp in length and is shown in Figure 53.
  • the pAAV-HBA-eGFP-P2A-5’mOTOF.SD vector includes an AAV ITR (SEQ ID NO: 59), a CMV enhancer (SEQ ID NO: 70), a sequence encoding a portion of 5’mOTOF (SEQ ID NO: 94), and a splice donor sequence (SEQ ID NO: 64).
  • the vector is pAAV-SA-3’mOTOF.WPRE (SEQ ID NO: 88), or is a vector that includes a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 88.
  • the pAAV-SA-3’mOTOF.WPRE vector is 4,674 bp in length and is shown in Figure 54.
  • the pAAV-SA-3’mOTOF.WPRE vector includes an AAV ITR (SEQ ID NO: 59), a splice acceptor sequence (SEQ ID NO: 65), a sequence encoding a portion 3’ mOTOF (SEQ ID NO: 95), a WPRE sequence (SEQ ID NO: 69), a BGHpA sequence (SEQ ID NO: 68), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-HBA-eGFP-P2A-5 , mOTOF.SD-AK, shown in Figure 55, and is 4,549 bp in length.
  • the pAAV-HBA-eGFP-P2A-5’mOTOF.SD-AK vector includes an AAV ITR (SEQ ID NO: 59), a CMV enhancer (SEQ ID NO: 70), a sequence encoding a portion 5’ mOTOF (SEQ ID NO: 94), a SD-intron sequence (SEQ ID NO: 72), an AK sequence (SEQ ID NO: 67), and an AAV ITR (SEQ ID NO: 60).
  • the vector is p AAV- AK-SA-3’ mOTOF -WPRE, shown in Figure 56 and is 4,751 bp in length.
  • the pAAV- AK- S A-3 ’ mOTOF -WPRE vector includes an AAV ITR (SEQ ID NO: 59), an AK sequence (SEQ ID NO: 66), a sequence encoding a portion of 3’ mOTOF (SEQ ID NO: 95), a WPRE sequence (SEQ ID NO: 69), a BGHpA sequence (SEQ ID NO: 68), and an AAV ITR (SEQ ID NO: 60).
  • the vector is pAAV-CMV-5’hOTOF-SD-AK (pl08 plasmid), shown in Figure 57 and is 4,567 bp in length.
  • the pAAV-CMV-5’hOTOF-SD-AK vector includes an AAV ITR (SEQ ID NO: 59), a FVII stuffer (SEQ ID NO: 90), a FVII stuffer (SEQ ID NO: 91), a CMV enhancer and promoter (SEQ ID NO: 70), a portion of a sequence encoding 5’hOTOF (SEQ ID NO: 62), a SD intron sequence (SEQ ID NO: 72), an AK sequence (SEQ ID NO: 67), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-HBA-5’hOTOF-SD-AP, shown in Figure 58, and is 4,540 bp in length.
  • the pAAV-HBA-5’hOTOF-SD-AP includes an AAV ITR sequence (SEQ ID NO: 59), a CMV enhancer (SEQ ID NO: 70), a sequence encoding a portion 5’hOTOF (SEQ ID NO: 62), an AP rec sequence (SEQ ID NO: 89), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-HBA-5’hOTOF-SD-AK, shown in Figure 59, and is 4,745 bp in length.
  • the pAAV-HBA-5’hOTOF-SD-AK includes an AAV ITR sequence (SEQ ID NO: 59), a FVIII stuffer (4677-5173) sequence (SEQ ID NO: 90), a FVIII stuffer (3679-4177) sequence (SEQ ID: 91), a CMV enhancer (SEQ ID NO: 70), a sequence encoding a portion 5’hOTOF (SEQ ID NO: 62), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector includes a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 90, or a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 91.
  • the vector is pAAV-HBA-5’hOTOFcodop-SD-AK, shown in Figure 60, and is 4,745 bp in length.
  • the pAAV- CMV-5’hOTOF-SD-AK vector includes an AAV ITR sequence (SEQ ID NO: 59), a FVIII stuffer (4677-5173) sequence (SEQ ID NO: 90), a FVIII stuffer (3679- 4177) sequence (SEQ ID: 91), a CMV enhancer (SEQ ID NO: 70), a sequence encoding a portion a 5’OTOF codop sequence (SEQ ID NO: 92), an AK sequence (SEQ ID NO: 67), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-HBA-5’hOTOFcodop-SD, shown in Figure 61, and is 4,668 bp in length.
  • the pAAV- HBA-5’hOTOFcodop-SD vector includes an AAV ITR sequence (SEQ ID NO: 59), a FVIII stuffer (4677-5173) sequence (SEQ ID NO: 90), a FVIII stuffer (3679-4177) sequence (SEQ ID: 91), a CMV enhancer (SEQ ID NO: 70), a sequence encoding a portion 5’OTOF codop sequence (SEQ ID NO: 92), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-CMV-5’hOTOF-SD, shown in Figure 62, and is 4,490 bp in length.
  • the pAAV- CMV-5’hOTOF-SD vector includes an AAV ITR sequence (SEQ ID NO: 59), two FVIII stuffer sequences, a CMV enhancer and promoter (SEQ ID NO: 70), a sequence encoding a portion of a 5’OTOF codop sequence (SEQ ID NO: 92), a SD-intron sequence (SEQ ID NO: 64), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-CMV-5’hOTOF-SD-AK, shown in Figure 63, and is 4,567 bp in length.
  • the p AAV-CM V-5 ’ hOTOF - SD- AK vector includes an AAV ITR sequence (SEQ ID NO:
  • the vector is pAAV-CBA-5’hOTOF-SD-AK, shown in Figure 64, and is 4,711 bp in length.
  • the pAAV-CBA-5’hOTOF-SD-AK vector includes an AAV ITR sequence (SEQ ID NO:
  • a CMV enhancer and chicken beta-actin promoter SEQ ID NO: 61
  • a chimeric intronic sequence a sequence encoding a portion of a 5’ hOTOF codop sequence (SEQ ID NO: 92), an intronic sequence, an AK sequence (SEQ ID NO: 67), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-CBA-5’hOTOF-SD, shown in Figure 65, and is 4,634 bp in length.
  • the pAAV- CBA-5’hOTOF-SD vector includes an AAV ITR sequence (SEQ ID NO: 59), a CMV enhancer and beta-actin promoter (SEQ ID NO: 61), a chimeric intronic sequence, a sequence encoding a portion of a 5’ OTOF codop sequence (SEQ ID NO: 92), an intronic sequence, and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-SA-3’OTOF, shown in Figure 66, and is 4,587 bp in length.
  • the pAAV-CBA- 5’hOTOF-SD vector includes an AAV ITR sequence (SEQ ID NO: 59), a splice acceptor sequence (SEQ ID NO: 65), a sequence encoding a portion 3OTOF sequence (SEQ ID NO: 63), a bGHpA sequence (SEQ ID NO: 68), a FVIII stuffer 9 sequence (SEQ ID NO: 57), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-AP-SA-3’OTOF, shown in Figure 67, and is 4,959 bp in length.
  • the pAAV-AP- SA-3OTOF vector includes an AAV ITR sequence (SEQ ID NO: 59), an AP rec sequence (SEQ ID NO: 89), a splice acceptor sequence (SEQ ID NO: 65), a sequence encoding a portion of a 3’OTOF sequence (SEQ ID NO: 63), a bGHpolyA sequence (SEQ ID NO: 68), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vector is pAAV-AK-SA-3’OTOFcodop, shown in Figure 68, and is 4,664 bp in length.
  • the p AAV - AK- S A-3 ’ OTOF codop vector includes an AAV ITR (SEQ ID NO: 59), an AK sequence (SEQ ID NO: 66), a sequence encoding a portion of a 3 OTOF codop sequence (SEQ ID NO: 93), a bGH polyA sequence (SEQ ID NO: 68), and an AAV ITR sequence (SEQ ID NO: 60).
  • the vectors provided herein can be of different sizes.
  • the choice of vector that is used in any of the compositions, kits, and methods described herein may depend on the size of the vector.
  • the vector(s) is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb.
  • the vector(s) is a plasmid and can have a total length in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, about 1 kb to about 15 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 k
  • the vector(s) is a transposon (e.g., PiggyBac transposon) and can include greater than 200 kb.
  • the vector(s) is a transposon having a total length in the range of about 1 kb to about 10 kb, about 1 kb to about 20 kb, about 1 kb to about 30 kb, about 1 kb to about 40 kb, about 1 kb to about 50 kb, about 1 kb to about 60 kb, about 1 kb to about 70 kb, about 1 kb to about 80 kb, about 1 kb to about 90 kb, about 10 kb to about 20 kb, about 10 kb to about 30 kb, about 10 kb to about 40 kb, about 10 kb to about 50 kb, about 10 kb to about 60 kb, about 10 kb to about 70 kb, about 10 kb to about 90 kb, about 10 k
  • 90 kb about 80 kb to about 100 kb, about 90 kb to about 100 kb, about 1 kb to about 100 kb, about 100 kb to about 200 kb, about 100 kb to about 300 kb, about 100 kb to about 400 kb, or about 100 kb to about 500 kb.
  • the vector is a cosmid and can have a total length of up to 55 kb.
  • the vector is a cosmid and has a total number of nucleotides of about 1 kb to about 10 kb, about 1 kb to about 20 kb, about 1 kb to about 30 kb, about 1 kb to about 40 kb, about 1 kb to about 50 kb, about 1 kb to about 55 kb, about 10 kb to about 20 kb, about 10 kb to about 30 kb, about 10 kb to about 40 kb, about 10 kb to about 50 kb, about 10 kb to about 55 kb, about 15 kb to about 55 kb, about 15 kb to about 15 kb to about 15 kb to about 15 kb, about 15 kb to about
  • the vector(s) is an artificial chromosome and can have a total number of nucleotides of about 100 kb to about 2000 kb.
  • the artificial chromosome(s) is a human artificial chromosome (HAC) and can have a total number of nucleotides in the range of about 1 kb to about 10 kb, 1 kb to about 20 kb, about 1 kb to about 30 kb, about 1 kb to about 40 kb, about 1 kb to about 50 kb, about 1 kb to about 60 kb, about 10 kb to about 20 kb, about 10 kb to about 30 kb, about 10 kb to about 40 kb, about 10 kb to about 50 kb, about 10 kb to about 60 kb, about 20 kb to about 30 kb, about 20 kb to about 40 kb, about 10 kb to about 50 kb, about 10 kb to about 60
  • the artificial chromosome(s) is a yeast artificial chromosome (YAC) and can have a total number of nucleotides up to 1000 kb.
  • the artificial chromosome(s) is a YAC having a total number of nucleotides in the range of about 100 kb to about 1,000 kb, about 100 kb to about 900 kb, about 100 kb to about 800 kb, about 100 kb to about 700 kb, about 100 kb to about 600 kb, about 100 kb to about 500 kb, about 100 kb to about 400 kb, about 100 kb to about 300 kb, about 100 kb to about 200 kb, about 200 kb to about 1,000 kb, about 200 kb to about 900 kb, about 200 kb to about 800 kb, about 200 kb to about 700 kb, about 200 kb to about 600 kb, about 200 kb to about 300 k
  • the artificial chromosome(s) is a bacterial artificial chromosome (BAC) and can have a total number of nucleotides of up to 750 kb.
  • the artificial chromosome(s) is a BAC and can have a total number of nucleotides in the range of about 100 kb to about 750 kb, about 100 kb to about 700 kb, about 100 kb to about 600 kb, about 100 kb to about 500 kb, about 100 kb to about 400 kb, about 100 kb to about 300 kb, about 100 kb to about 200 kb, about 150 kb to about 750 kb, about 150 kb to about 700 kb, about 150 kb to about 600 kb, about 150 kb to about 500 kb, about 150 kb to about 400 kb, about 150 kb to about 300 kb, about 150 kb to about 200 kb, about 150 kb to about
  • the artificial chromosome(s) is a PI -derived artificial chromosome (PAC) and can have a total number of nucleotides of up to 300 kb.
  • the PI -derived artificial chromosome(s) can have a total number of nucleotides in the range of about 100 kb to about 300 kb, about 100 kb to about 200 kb, or about 200 kb to about 300 kb.
  • the vector(s) is a viral vector and can have a total number of nucleotides of up to 10 kb. In some embodiments, the viral vector(s) can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about
  • the vector(s) is a lentivirus and can have a total number of nucleotides of up to 8 kb.
  • the lentivirus(es) can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 2 kb
  • the vector(s) is an adenovirus and can have a total number of nucleotides of up to 8 kb.
  • the adenovirus(es) can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about 3 kb to about 4 kb, about
  • the vector(s) is an adeno-associated virus (AAV vector) and can include a total number of nucleotides of up to 5 kb.
  • AAV vector(s) can include a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.
  • the vector(s) is a Gateway® vector and can include a total number of nucleotides of up to 5 kb.
  • each Gateway® vector(s) includes a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.
  • the at least two different vectors can be substantially the same type of vector and may differ in size. In some embodiments, the at least two different vectors can be different types of vector, and may have substantially the same size or have different sizes.
  • any of the at least two vectors can have a total number of nucleotides in the range of about 500 nucleotides to about 10,000 nucleotides, about 500 nucleotides to about 9,500 nucleotides, about 500 nucleotides to about 9,000 nucleotides, about 500 nucleotides to about 8,500 nucleotides, about 500 nucleotides to about 8,000 nucleotides, about 500 nucleotides to about 7,800 nucleotides, about 500 nucleotides to about 7,600 nucleotides, about 500 nucleotides to about 7,400 nucleotides, about 500 nucleotides to about 7,200 nucleotides, about 500 nucleotides to about 7,000 nucleotides, about 500 nucleotides to about 6,800 nucleotides, about 500 nucleotides to about 6,600 nucleotides, about 500 nucleotides to about 6,400 nucleotides, about 500 nucleotides to about 500
  • 1,200 nucleotides to about 7,000 nucleotides about 1,200 nucleotides to about 6,800 nucleotides, about 1,200 nucleotides to about 6,600 nucleotides, about 1,200 nucleotides to about 6,400 nucleotides, about 1,200 nucleotides to about 6,200 nucleotides, about
  • 1,200 nucleotides to about 6,000 nucleotides about 1,200 nucleotides to about 5,800 nucleotides, about 1,200 nucleotides to about 5,600 nucleotides, about 1,200 nucleotides to about 5,400 nucleotides, about 1,200 nucleotides to about 5,000 nucleotides, about
  • 1,200 nucleotides to about 4,800 nucleotides about 1,200 nucleotides to about 4,600 nucleotides, about 1,200 nucleotides to about 4,400 nucleotides, about 1,200 nucleotides to about 4,200 nucleotides, about 1,200 nucleotides to about 4,000 nucleotides, about
  • 1,200 nucleotides to about 3,800 nucleotides about 1,200 nucleotides to about 3,600 nucleotides, about 1,200 nucleotides to about 3,400 nucleotides, about 1,200 nucleotides to about 3,200 nucleotides, about 1,200 nucleotides to about 3,000 nucleotides, about 1,200 nucleotides to about 2,800 nucleotides, about 1,200 nucleotides to about 2,600 nucleotides, about 1,200 nucleotides to about 2,400 nucleotides, about 1,200 nucleotides to about 2,200 nucleotides, about 1,200 nucleotides to about 2,000 nucleotides, about 1,200 nucleotides to about 1,800 nucleotides, about 1,200 nucleotides to about 1,600 nucleotides, about 1,200 nucleotides to about 1,400 nucleotides, about 1,400 nucleotides to about 10,000 nucleotides, about 1,400 nucle
  • 1,400 nucleotides to about 7,400 nucleotides about 1,400 nucleotides to about 7,200 nucleotides, about 1,400 nucleotides to about 7,000 nucleotides, about 1,400 nucleotides to about 6,800 nucleotides, about 1,400 nucleotides to about 6,600 nucleotides, about
  • 1,400 nucleotides to about 6,400 nucleotides about 1,400 nucleotides to about 6,200 nucleotides, about 1,400 nucleotides to about 6,000 nucleotides, about 1,400 nucleotides to about 5,800 nucleotides, about 1,400 nucleotides to about 5,600 nucleotides, about
  • 1,400 nucleotides to about 5,400 nucleotides about 1,400 nucleotides to about 5,200 nucleotides, about 1,400 nucleotides to about 5,000 nucleotides, about 1,400 nucleotides to about 4,800 nucleotides, about 1,400 nucleotides to about 4,600 nucleotides, about
  • 1,400 nucleotides to about 4,400 nucleotides about 1,400 nucleotides to about 4,200 nucleotides, about 1,400 nucleotides to about 4,000 nucleotides, about 1,400 nucleotides to about 3,800 nucleotides, about 1,400 nucleotides to about 3,600 nucleotides, about
  • 1,400 nucleotides to about 3,400 nucleotides about 1,400 nucleotides to about 3,200 nucleotides, about 1,400 nucleotides to about 3,000 nucleotides, about 1,400 nucleotides to about 2,600 nucleotides, about 1,400 nucleotides to about 2,400 nucleotides, about
  • 1,600 nucleotides to about 6,400 nucleotides about 1,600 nucleotides to about 6,200 nucleotides, about 1,600 nucleotides to about 6,000 nucleotides, about 1,600 nucleotides to about 5,800 nucleotides, about 1,600 nucleotides to about 5,600 nucleotides, about
  • 1,600 nucleotides to about 5,400 nucleotides about 1,600 nucleotides to about 5,200 nucleotides, about 1,600 nucleotides to about 5,000 nucleotides, about 1,600 nucleotides to about 4,800 nucleotides, about 1,600 nucleotides to about 4,600 nucleotides, about
  • 1,600 nucleotides to about 4,400 nucleotides about 1,600 nucleotides to about 4,200 nucleotides, about 1,600 nucleotides to about 4,000 nucleotides, about 1,600 nucleotides to about 3,800 nucleotides, about 1,600 nucleotides to about 3,600 nucleotides, about
  • 1,600 nucleotides to about 3,400 nucleotides about 1,600 nucleotides to about 3,200 nucleotides, about 1,600 nucleotides to about 3,000 nucleotides, about 1,600 nucleotides to about 2,800 nucleotides, about 1,600 nucleotides to about 2,600 nucleotides, about
  • 1,600 nucleotides to about 2,400 nucleotides about 1,600 nucleotides to about 2,200 nucleotides, about 1,600 nucleotides to about 2,000 nucleotides, about 1,600 nucleotides to about 1,800 nucleotides, about 1,800 nucleotides to about 10,000 nucleotides, about
  • 1,800 nucleotides to about 9,500 nucleotides about 1,800 nucleotides to about 9,000 nucleotides, about 1,800 nucleotides to about 8,500 nucleotides, about 1,800 nucleotides to about 8,000 nucleotides, about 1,800 nucleotides to about 7,800 nucleotides, about
  • 2,400 nucleotides to about 7,400 nucleotides about 2,400 nucleotides to about 7,200 nucleotides, about 2,400 nucleotides to about 7,000 nucleotides, about 2,400 nucleotides to about 6,800 nucleotides, about 2,400 nucleotides to about 6,600 nucleotides, about
  • 2,400 nucleotides to about 6,400 nucleotides about 2,400 nucleotides to about 6,200 nucleotides, about 2,400 nucleotides to about 6,000 nucleotides, about 2,400 nucleotides to about 5,800 nucleotides, about 2,400 nucleotides to about 5,600 nucleotides, about
  • 2,400 nucleotides to about 5,400 nucleotides about 2,400 nucleotides to about 5,200 nucleotides, about 2,400 nucleotides to about 5,000 nucleotides, about 2,400 nucleotides to about 4,800 nucleotides, about 2,400 nucleotides to about 4,600 nucleotides, about
  • 2,400 nucleotides to about 4,400 nucleotides about 2,400 nucleotides to about 4,200 nucleotides, about 2,400 nucleotides to about 4,000 nucleotides, about 2,400 nucleotides to about 3,800 nucleotides, about 2,400 nucleotides to about 3,600 nucleotides, about
  • 2,600 nucleotides to about 6,800 nucleotides about 2,600 nucleotides to about 6,600 nucleotides, about 2,600 nucleotides to about 6,400 nucleotides, about 2,600 nucleotides to about 6,200 nucleotides, about 2,600 nucleotides to about 6,000 nucleotides, about
  • 2,600 nucleotides to about 5,800 nucleotides about 2,600 nucleotides to about 5,600 nucleotides, about 2,600 nucleotides to about 5,400 nucleotides, about 2,600 nucleotides to about 5,200 nucleotides, about 2,600 nucleotides to about 5,000 nucleotides, about
  • 2,600 nucleotides to about 4,800 nucleotides about 2,600 nucleotides to about 4,600 nucleotides, about 2,600 nucleotides to about 4,400 nucleotides, about 2,600 nucleotides to about 4,200 nucleotides, about 2,600 nucleotides to about 4,000 nucleotides, about
  • 2,600 nucleotides to about 3,800 nucleotides about 2,600 nucleotides to about 3,600 nucleotides, about 2,600 nucleotides to about 3,400 nucleotides, about 2,600 nucleotides to about 3,200 nucleotides, about 2,600 nucleotides to about 3,000 nucleotides, about
  • 2,600 nucleotides to about 2,800 nucleotides about 2,800 nucleotides to about 10,000 nucleotides, about 2,800 nucleotides to about 9,500 nucleotides, about 2,800 nucleotides to about 9,000 nucleotides, about 2,800 nucleotides to about 8,500 nucleotides, about
  • 2,800 nucleotides to about 8,000 nucleotides about 2,800 nucleotides to about 7,800 nucleotides, about 2,800 nucleotides to about 7,600 nucleotides, about 2,800 nucleotides to about 7,400 nucleotides, about 2,800 nucleotides to about 7,200 nucleotides, about
  • 2,800 nucleotides to about 7,000 nucleotides about 2,800 nucleotides to about 6,800 nucleotides, about 2,800 nucleotides to about 6,600 nucleotides, about 2,800 nucleotides to about 6,400 nucleotides, about 2,800 nucleotides to about 6,200 nucleotides, about
  • 2,800 nucleotides to about 6,000 nucleotides about 2,800 nucleotides to about 5,800 nucleotides, about 2,800 nucleotides to about 5,600 nucleotides, about 2,800 nucleotides to about 5,400 nucleotides, about 2,800 nucleotides to about 5,200 nucleotides, about
  • 2,800 nucleotides to about 5,000 nucleotides about 2,800 nucleotides to about 4,800 nucleotides, about 2,800 nucleotides to about 4,600 nucleotides, about 2,800 nucleotides to about 4,400 nucleotides, about 2,800 nucleotides to about 4,200 nucleotides, about
  • 3,200 nucleotides to about 4,800 nucleotides about 3,200 nucleotides to about 4,600 nucleotides, about 3,200 nucleotides to about 4,400 nucleotides, about 3,200 nucleotides to about 4,200 nucleotides, about 3,200 nucleotides to about 4,000 nucleotides, about 3,200 nucleotides to about 3,800 nucleotides, about 3,200 nucleotides to about 3,600 nucleotides, about 3,200 nucleotides to about 3,400 nucleotides, about 3,400 nucleotides to about 10,000 nucleotides, about 3,400 nucleotides to about 9,500 nucleotides, about
  • 3,400 nucleotides to about 5,400 nucleotides about 3,400 nucleotides to about 5,200 nucleotides, about 3,400 nucleotides to about 5,000 nucleotides, about 3,400 nucleotides to about 4,800 nucleotides, about 3,400 nucleotides to about 4,600 nucleotides, about
  • 3,600 nucleotides to about 7,800 nucleotides about 3,600 nucleotides to about 7,600 nucleotides, about 3,600 nucleotides to about 7,400 nucleotides, about 3,600 nucleotides to about 7,200 nucleotides, about 3,600 nucleotides to about 7,000 nucleotides, about
  • 3,600 nucleotides to about 6,800 nucleotides about 3,600 nucleotides to about 6,600 nucleotides, about 3,600 nucleotides to about 6,400 nucleotides, about 3,600 nucleotides to about 6,200 nucleotides, about 3,600 nucleotides to about 6,000 nucleotides, about
  • 3,600 nucleotides to about 5,800 nucleotides about 3,600 nucleotides to about 5,600 nucleotides, about 3,600 nucleotides to about 5,400 nucleotides, about 3,600 nucleotides to about 5,200 nucleotides, about 3,600 nucleotides to about 5,000 nucleotides, about 3,600 nucleotides to about 4,800 nucleotides, about 3,600 nucleotides to about 4,600 nucleotides, about 3,600 nucleotides to about 4,400 nucleotides, about 3,600 nucleotides to about 4,200 nucleotides, about 3,600 nucleotides to about 4,000 nucleotides, about 3,600 nucleotides to about 3,800 nucleotides, about 3,800 nucleotides to about 10,000 nucleotides, about 3,800 nucleotides to about 9,500 nucleotides, about 3,800 nucleotides to about 9,000 nucleotides, about 3,800 nucle
  • Non limiting examples of methods for introducing nucleic acid into a mammalian cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.
  • lipofection e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposome
  • any of the vectors described herein can be introduced into a mammalian cell by, for example, lipofection.
  • Various molecular biology techniques that can be used to introduce a mutation(s) and/or a deletion(s) into an endogenous gene are also known in the art.
  • Non-limiting examples of such techniques include site-directed mutagenesis, CRISPR (e.g., CRISPR/Cas9-induced knock-in mutations and CRISPR/Cas9-induced knock-out mutations), and TALENs. These methods can be used to correct the sequence of a defective endogenous gene present in a chromosome of a target cell.
  • any of the vectors described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly A) sequence, and a Kozak consensus sequence.
  • a control sequence e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly A) sequence, and a Kozak consensus sequence.
  • a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and/or a tissue-specific promoter.
  • promoters are described herein. Additional examples of promoters are known in the art.
  • a vector encoding an N-terminal portion of an otoferlin protein can include a promoter and/or an enhancer.
  • the vector encoding the N-terminal portion of the otoferlin protein can include any of the promoters and/or enhancers described herein or known in the art.
  • the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art.
  • the promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter.
  • the promoter is a RNA polymerase III promoter, including, but not limited to, a HI promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter.
  • the promoter will generally be one that is able to promote transcription in cochlear cells such as hair cells.
  • the promoter is a cochlea-specific promoter or a cochlea-oriented promoter.
  • promoters are known in the art that can be used herein.
  • Non-limiting examples of promoters that can be used herein include: human elongation factor la- subunit (EFla) (Liu et al. (2007) Exp. Mol. Med. 39(2): 170-175; Accession No. J04617.1; Gill et al., Gene Ther. 8(20): 1539-1546, 2001; Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332; Ikeda et al., Gene Ther. 9:932- 938. 2002: Gilham et al., J Gene Med.
  • EFla human elongation factor la- subunit
  • cytomegalovirus Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332; Gray et al., Human Gene Ther. 22:1143-1153, 2011
  • CMV human immediate-early cytomegalovirus
  • UBC human ubiquitin C
  • mice phosphoglycerate kinase 1 polyoma adenovirus, simian virus 40 (SV40), b-globin, b- actin, a-fetoprotein, g-globin, b-interferon, g-glutamyl transferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3 -phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g.
  • human T-cell leukemia virus HTLV human T-cell leukemia virus HTLV
  • AAV ITR interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and GRP94), a-2-macroglobulin, vimentin, MHC class I gene H-2K b, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone a gene, immunoglobulin light chain, T-cell receptor, HLA DQa and ⁇ 3b, interleukin-2 receptor, MHC class II, MHC class II HLA-DRa, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (S
  • HBA human b-actin promoter
  • murine myosin VIIA murine myosin VIIA
  • human myosin VIIA human myosin VIIA
  • hsMyo7 human myosin VIIA
  • NG_009086.1 murine poly(ADP- ribose) polymerase 2
  • musPARP2 murine poly(ADP- ribose) polymerase 2
  • promoter Human Gene Ther. 13(7):829-840, 2002; Cunningham et al ,Mol. Ther. 16(6): 1081- 1088, 2008), and a CBA hybrid (CBh) (Gray et al. (2011) Hum. Gen. Therapy 22: 1143- 1153; Accession No. KF926476.1 or KC152483.1) .
  • CBA hybrid CBA hybrid
  • Additional examples of promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology, Freeman and Company, New York 2007.
  • the promoter is the CMV immediate early promoter.
  • the promoter is a CAG promoter or a CAG/CBA promoter.
  • a vector or construct of the present disclosure comprises a CAG promoter.
  • a CAG promoter comprises, in order from 5’ to 3’, the nucleotide sequences of SEQ ID NOs: 98, 99, and 100.
  • a CAG promoter comprises a CMV early enhancer element (e.g., SEQ ID NO: 98), a chicken beta actin (CBA) gene sequence (e.g., SEQ ID NO: 99), and a chimeric intron/3’ splice sequence from the rabbit beta globin gene (e.g., SEQ ID NO: 100).
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • RNA refers to a nucleotide sequence that, when operably linked with a nucleic acid encoding a protein (e.g., an otoferlin protein), causes RNA to be transcribed from the nucleic acid in a mammalian cell under most or all physiological conditions.
  • a protein e.g., an otoferlin protein
  • constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al. Cell 41:521-530, 1985), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 -alpha promoter (Invitrogen).
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter the dihydrofolate reductase promoter
  • beta-actin promoter the beta-actin promoter
  • PGK phosphoglycerol kinase
  • EF1 -alpha promoter Invitrogen
  • inducible promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Additional examples of inducible promoters are known in the art.
  • inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al. Proc. Natl. Acad. Sci. U.S.A. 93:3346-3351, 1996), the tetracycline-repressible system (Gossen et al. Proc. Natl. Acad. Sci. U.S.A.
  • tissue-specific promoter refers to a promoter that is active only in certain specific cell types and/or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory proteins that bind to the tissue-specific promoter).
  • tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • regulatory sequence refers to a nucleic acid sequence which is regulates expression of a gene product operably linked to the regulatory sequence. In some instances, this sequence may be an enhancer sequence and other regulatory elements which regulate expression of the gene product.
  • the regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.
  • the tissue-specific promoter is a cochlea-specific promoter. In some embodiments, the tissue-specific promoter is a cochlear hair cell-specific promoter.
  • cochlear hair cell-specific promoters include but are not limited to: a ATOH1 promoter, a POU4F3 promoter, a LHX3 promoter, a MY07A promoter, a MY06 promoter, a a9ACHR promoter, and a alOACHR promoter.
  • a vector can include a promoter sequence and/or an enhancer sequence.
  • the term “enhancer” refers to a nucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., an otoferlin protein). Enhancer sequences (50-1500 base pairs in length) generally increase the level of transcription by providing additional binding sites for transcription-associated proteins (e.g., transcription factors). In some embodiments, an enhancer sequence is found within an intronic sequence. Unlike promoter sequences, enhancer sequences can act at much larger distance away from the transcription start site (e.g., as compared to a promoter).
  • Non-limiting examples of enhancers include a RSV enhancer, a CMV enhancer, and a SV40 enhancer.
  • An example of a CMV enhancer is described in, e.g., Boshart et ah, Cell 41(2):521-530, 1985.
  • a 5' cap also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m.sup.7G cap
  • the 5' cap consists of a terminal group which is linked to the first transcribed nucleotide.
  • any of the vectors provided herein can include a poly(A) sequence.
  • Most nascent eukaryotic mRNAs possess a poly(A) tail at their 3’ end which is added during a complex process that includes cleavage of the primary transcript and a coupled polyadenylation reaction (see, e.g., Proudfoot et ah, Cell 108:501-512, 2002).
  • the poly(A) tail confers mRNA stability and transferability (Molecular Biology of the Cell, Third Edition by B. Alberts et ah, Garland Publishing, 1994).
  • the poly(A) sequence is positioned 3’ to the nucleic acid sequence encoding the C-terminus of the otoferlin protein.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • the 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site.
  • adenosine residues are added to the free 3' end at the cleavage site.
  • a “poly (A) signal sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the additional of a series of adenosines to the 3’ end of the cleaved mRNA.
  • a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA.
  • the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400.
  • Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
  • poly(A) signal sequences that can be used, including those derived from bovine growth hormone (bgh) (Woychik et al., Proc. Natl. Acad. Sci. U.S.A. 81(13):3944-3948, 1984; U.S. Patent No. 5,122,458; Yew et al., Human Gene Ther. 8(5):575-584, 1997; Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332, 2001; Wu et al., Mol. Ther. 16(2):280-289, 2008; Gray et al., Human Gene Ther.
  • bovine growth hormone bgh
  • a non-limiting example of a poly(A) signal sequence is SEQ ID NO: 68, 76, or 77.
  • the poly(A) signal sequence can be the sequence AATAAA.
  • the AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA which are capable of signaling polyadenylation, including ATTAAA, AGTAAA, CAT AAA, TAT A A A, GAT A A A, ACT AAA, A AT AT A, AAGAAA,
  • a AT AG A, AATTAA, or A AT A AG see, e.g., WO 06/12414.
  • the poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., the pCl-neo expression vector of Promega which is based on Levitt el al, Genes Dev. 3(7): 1019-1025, 1989).
  • the poly(A) signal sequence is the polyadenylation signal of soluble neuropilin-1 (sNRP) (AAATAAAATACGAAATG) (see, e.g., WO 05/073384).
  • a poly(A) sequence is a bovine growth hormone poly(A) sequence.
  • a bGH poly(A) sequence comprises or is the sequence of SEQ ID NO:
  • a vector or construct of the present disclosure comprises a boving growth hormone polyA sequence represented by SEQ ID NO: 108. Additional examples of poly(A) signal sequences are known in the art.
  • any of the vectors provided herein can include a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), e.g., SEQ ID NO: 69.
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • a vector encoding the C-terminus of the otoferlin protein can include a polynucleotide internal ribosome entry site (IRES).
  • IRES polynucleotide internal ribosome entry site
  • An IRES sequence is used to produce more than one polypeptide from a single gene transcript.
  • An IRES forms a complex secondary structure that allows translation initiation to occur from any position with an mRNA immediately downstream from where the IRES is located (see, e.g., Pelletier and Sonenb erg, Mol. Cell. Biol. 8(3): 1103-1112, 1988).
  • IRES sequences known to those skilled in the art, including those from, e.g., foot and mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricket paralysis virus, human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis C virus (HCV), and poliovirus (PV).
  • FMDV foot and mouth disease virus
  • EMCV encephalomyocarditis virus
  • HRV human rhinovirus
  • HCV human immunodeficiency virus
  • HAV hepatitis A virus
  • HCV hepatitis C virus
  • PV poliovirus
  • the IRES sequence that is incorporated into the vector that encodes the C-terminus of an otoferlin protein is the foot and mouth disease virus (FMDV).
  • FMDV foot and mouth disease virus
  • the Foot and Mouth Disease Virus 2A sequence is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins (Ryan, M D et ak, EMBO 4:928-933, 1994; Mattion et al., ./. Virology 70:8124-8127, 1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et ak,
  • any of the vectors provided herein can optionally include a sequence encoding a reporter protein (“a reporter sequence”).
  • reporter sequences Non-limiting examples of reporter sequences are described herein. Additional examples of reporter sequences are known in the art.
  • the reporter sequence can be used to verify the tissue-specific targeting capabilities and tissue-specific promoter regulatory activity of any of the vectors described herein.
  • a NTF3 protein can have a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 78.
  • a NTF3 protein can include a sequence that is identical to SEQ ID NO: 78, except that it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions and/or deletions.
  • a NTF3 protein can be encoded by a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 79 or 80.
  • amino acids that are not conserved between the same protein from different species is less likely to have an effect on the function of a protein and therefore, these amino acids should be selected for mutation.
  • Amino acids that are conserved between the same protein from different species should not be mutated, as these mutations are more likely to result in a change in the function of the protein.
  • Non-limiting examples of neutrophin-3 from other mammalian species are shown below.
  • Cow Neurotrophin-3 (SEQ ID NO: 81)
  • Rat Neurotrophin-3 (SEQ ID NO: 82)
  • Pig Neurotrophin-3 (SEQ ID NO: 83) Flanking Regions Untranslated Regions (UTRs)
  • any of the vectors described herein can include an untranslated region.
  • a vector can includes a 5’ UTR or a 3’ UTR.
  • Untranslated regions (UTRs) of a gene are transcribed but not translated.
  • the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon.
  • the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the regulatory features of a UTR can be incorporated into any of the vectors, compositions, kits, or methods as described herein to enhance the stability of an otoferlin protein.
  • Natural 5' UTRs include a sequence that plays a role in translation initiation. They harbor signatures like Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), which is followed by another “G”. The 5' UTRs have also been known, e.g., to form secondary structures that are involved in elongation factor binding.
  • a 5’ UTR is included in any of the vectors described herein.
  • Non-limiting examples of 5’ UTRs including those from the following genes: albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as a mRNA.
  • a 5’ UTR from a mRNA that is transcribed by a cell in the cochlea can be included in any of the vectors, compositions, kits, and methods described herein.
  • AU-rich elements can be separated into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs.
  • Class II AREs possess two or more overlapping UUAUUUA(U/A) (U/A) nonamers.
  • GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs.
  • Class III AREs are less well defined. These U- rich regions do not contain an AUUUA motif. Two well-studied examples of this class are c-Jun and myogenin mRNAs.
  • HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
  • the introduction, removal, or modification of 3' UTR AREs can be used to modulate the stability of an mRNA encoding an otoferlin protein.
  • AREs can be removed or mutated to increase the intracellular stability and thus increase translation and production of an otoferlin protein.
  • non-UTR sequences may be incorporated into the 5' or 3' UTRs.
  • introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the vectors, compositions, kits, and methods provided herein. Incorporation of intronic sequences may increase protein production as well as mRNA levels.
  • An intron can be an intron from an otoferlin gene or can be an intron from a heterologous gene, e.g., a hybrid adenovirus/mouse immunoglobulin intron (Yew et al., Human Gene Ter.
  • Non-limiting examples of a splice donor and splice acceptor sequences are SEQ ID NOs: 64 and 65, respectively; SEQ ID NOs: 72 and 73, respectively; and SEQ ID NOs: 74 and 75, respectively.
  • the splice donor sequence has the sequence of SEQ ID NO: 102.
  • a vector of construct of the present disclosure comprises a splice donor sequence of SEQ ID NO: 102.
  • the vector or construct comprising a splice donor sequence also comprises a 5’ portion of the OTOF gene or OTOF cDNA (e.g., SEQ ID NO: 101) upstream of the splice donor sequence.
  • the splice acceptor sequence has the sequence of SEQ ID NO: 106.
  • a vector or construct of the present disclosure comprises a splice acceptor sequence of SEQ ID NO: 106.
  • the vector or construct comprising a splice acceptor sequence also comprises a 3’ portion of the OTOF gene or OTOF cDNA (e.g., SEQ ID NO: 107) downstream of the splice acceptor sequence.
  • any of the vectors provided herein can optionally include a sequence encoding a destabilization domain (“a destabilization domain sequence”).
  • a destabilization domain is an amino acid sequence that decreases the in vivo or in vitro half-life of a protein that includes the destabilization domain, e.g., as compared to the same protein lacking the stabilization domain.
  • a destabilization domain may result in the targeting of a protein that includes the destabilization domain for proteosomal degradation.
  • Non limiting examples of destabilization domains include the destabilizing domain of the E. coli dihydrofolate reductase (DHFR) (Iwamoto et al. (2010) Chem. Biol.
  • SEQ ID NO: 53 is an exemplary amino acid sequence of a DHFR destabilization domain. Additional examples of destabilization domains are known in the art.
  • any of the vectors provided herein can optionally include a degradation sequence, e.g., a CL1 degradation sequence of SEQ ID NO: 71.
  • one or more vectors or constructs of the present disclosure comprise(s) one or more recombinogenic sequences.
  • a recombinogenic sequence is or comprises a portion of a gene sequence.
  • a recombinogenic sequence is derived from an alkaline phosphatase gene.
  • a recombinogenic sequence is derived from an FI phage.
  • a recombinogenic sequence is an AK sequence derived from an FI phage.
  • such an AK recombinogenic sequence is SEQ ID NO:
  • each of two vectors comprises a recombinogenic sequence.
  • a composition of the present disclosure comprises a first vector with a splice donor sequence (e.g., SEQ ID NO: 102) located downstream of a 5’ portion of the OTOF gene or OTOF cDNA (e.g., SEQ ID NO: 101) and upstream of an AK recombinogenic sequence (e.g., SEQ ID NO: 103) and a second vector with a splice acceptor sequence (e.g., SEQ ID NO: 106) located upstream of a 3’ portion of the OTOF gene or OTOF cDNA (e.g., SEQ ID NO: 107) and downstream of an AK recombinogenic sequence (e.g., SEQ ID NO: 103).
  • a splice donor sequence e.g., SEQ ID NO: 102
  • a splice acceptor sequence e.g., SEQ ID NO: 106
  • each vector can be designed to contain a total of about 4,000 base pairs to about 4,700 base pairs, e.g., about 4,000 base pairs to about 4,650 base pairs, about 4,000 base pairs to about 4,600 base pairs, about 4,000 base pairs to about 4,550 base pairs, about 4,000 base pairs to about 4,500 base pairs, about 4,000 base pairs to about 4,450 base pairs, about 4,000 base pairs to about 4,400 base pairs, about 4,000 base pairs to about 4,350 base pairs, about 4,000 base pairs to about 4,300 base pairs, about 4,000 base pairs to about 4,250 base pairs, about 4,000 base pairs to about 4,200 base pairs, about 4,000 base pairs to about 4,150 base pairs, about 4,000 base pairs to about 4,100 base pairs, about 4,000 base pairs to about 4,050 base pairs, about 4,050 base pairs to about 4,700 base pairs, about 4,050 base pairs
  • a stuffer sequence can be any nucleotide sequence, e.g., up to 1000 bp, that can be included in any of the vectors described herein that is not transcribed and that does not serve a regulatory function in order to achieve a desirable vector size (e.g., a vector size of about 4 kb to about 5 kb, or any of the vector sizes provided herein).
  • a stuffer sequence can be any nucleotide sequence of about 100 bp to about 1000 bp (e.g., about 100 bp to about 900 bp, about 100 bp to about 800 bp, about 100 bp to about 700 bp, about 100 bp to about 600 bp, about 100 bp to about 500 bp, about 100 bp to about 400 bp, about 100 bp to about 300 bp, about 100 bp to about 100 bp, about 200 bp to about 1000 bp, about 200 bp to about 900 bp, about 200 bp to about 800 bp, about 200 bp to about 700 bp, about 200 bp to about 600 bp, about 200 bp to about 500 bp, about 200 bp to about 400 bp, about 200 bp to about 300 bp, about 300 bp to about 1000 bp, about 300 bp to about 1000 bp, about 300 b
  • SEQ ID NOs. 54-58, 90 and 91 are exemplary human factor VIII stuffer sequences that can be used in any of the vectors described herein. Additional stuffer sequences are known in the art. Exemplary vectors that include stuffer sequences are shown in Figures 21-31, 36,37, 59-63 and 66.
  • compositions comprising one or more vectors to deliver a therapeutic gene, e.g., an entire therapeutic gene or a functional portion thereof, to a subject in need thereof.
  • a therapeutic gene e.g., an entire therapeutic gene or a functional portion thereof
  • the otoferlin gene is too large to be packaged into a single recombinant vector, e.g., a recombinant AAV vector.
  • two or more vectors are employed to deliver a therapeutic gene, e.g., an entire therapeutic gene to a subject in need thereof.
  • a dual vector system is used, wherein each of two vectors comprises a portion of the human otoferlin gene and, when delivered in vivo, the constructs come together to generate a polynucleotide that encodes a full length, functional, otoferlin protein.
  • one or more strategies is used, for example, (i) a concatemerization-trans-splicing strategy, (ii) a hybrid intronic-homologous recombination-trans-splicing strategy, and (iii) an exonic homologous recombination strategy, as summarized by Pryadkina et al. Meth Clin Devel 2015, 2:15009.
  • a cell e.g., a mammalian cell
  • a cell that includes any of the nucleic acids, vectors (e.g., at least two different vectors described herein), or compositions described herein.
  • the nucleic acids and vectors described herein can be introduced into any mammalian cell.
  • Non-limiting examples of vectors and methods for introducing vectors into mammalian cells are described herein.
  • the cell is a human cell, a mouse cell, a porcine cell, a rabbit cell, a dog cell, a cat cell, a rat cell, a sheep cell, a cat cell, a horse cell, or a non-human primate cell.
  • the cell is a specialized cell of the cochlea. In some embodiments, the cell is a cochlear inner hair cell or a cochlear outer hair cell. In some embodiments, the cell is a cochlear inner hair cell. In some embodiments, the cell is a cochlear inner hair cell.
  • the mammalian cell is in vitro. In some embodiments, the mammalian cell is present in a mammal. In some embodiments, the mammalian cell is obtained from a subject. In some embodiments, the mammalian cell is an autologous cell obtained from a subject and/or is cultured ex vivo.
  • Also provided herein is a method of introducing into a cochlea of a mammal (e.g., a human) a therapeutically effective amount of any of the compositions described herein. Also provided are methods of increasing expression of an active otoferlin protein (e.g., a full-length otoferlin protein) in an inner hair cell in a cochlea of a mammal (e.g., a human) that include introducing into the cochlea of the mammal a therapeutically effective amount of any of the compositions described herein.
  • an active otoferlin protein e.g., a full-length otoferlin protein
  • the methods described herein can further include administering a neurotrophic factor to a cochlea of a subject (e.g., at substantially the same time as or before, or after, any of the compositions described herein are administered to the subject).
  • the methods described herein can further include administering a cochlear implant to a subject (e.g., at substantially the same time as or before, or after, any of the compositions described herein are administered to the subject).
  • the mammal has been previously identified as having a defective otoferlin gene (e.g., an otoferlin gene having a mutation that results in a decrease in the expression and/or activity of an otoferlin protein encoded by the gene).
  • Some embodiments of any of these methods further include, prior to the introducing or administering step, determining that the subject has a defective otoferlin gene.
  • Some embodiments of any of these methods can further include detecting a mutation in an otoferlin gene in a subject.
  • Some embodiments of any of the methods can further include identifying or diagnosing a subject as having non-symptomatic sensorineural hearing loss.
  • two or more doses of any of the compositions described herein are introduced or administered into the cochlea of the mammal or subject.
  • Some embodiments of any of these methods can include introducing or administering a first dose of the composition into the cochlea of the mammal or subject, assessing hearing function of the mammal or subject following the introducing or the administering of the first dose, and administering an additional dose of the composition into the cochlea of the mammal or subject found not to have a hearing function within a normal range (e.g., as determined using any test for hearing known in the art).
  • the composition can be formulated for intra-cochlear administration. In some embodiments of any of the methods described herein, the compositions described herein can be administered via intra-cochlear administration or local administration. In some embodiments of any of the methods described herein, the compositions are administered through the use of a medical device (e.g., any of the exemplary medical devices described herein).
  • a medical device e.g., any of the exemplary medical devices described herein.
  • intra-cochlear administration can be performed using any of the methods described herein or known in the art.
  • a composition can be administered or introduced into the cochlea using the following surgical technique: first using visualization with a 0 degree, 2.5-mm rigid endoscope, the external auditory canal is cleared and a round knife is used to sharply delineate an approximately 5-mm tympanomeatal flap. The tympanomeatal flap is then elevated and the middle ear is entered posteriorly. The chorda tympani nerve is identified and divided, and a currette is used to remove the scutal bone, exposing the round window membrane.
  • a surgical laser may be used to make a small 2-mm fenestration in the oval window to allow for perilymph displacement during trans-round window membrane infusion of the composition.
  • the microinfusion device is then primed and brought into the surgical field.
  • the device is maneuvered to the round window, and the tip is seated within the bony round window overhang to allow for penetration of the membrane by the microneedle(s).
  • the footpedal is engaged to allow for a measured, steady infusion of the composition.
  • the device is then withdrawn and the round window and stapes foot plate are sealed with a gelfoam patch.
  • the present disclosure describes a delivery approach that utilizes a minimally invasive, well-accepted surgical technique for accessing the middle ear and/or inner ear through the external auditory canal.
  • the procedure includes opening one of the physical barriers between the middle and inner ear at the oval window, and subsequently using a device disclosed herein, e.g., as shown in FIGs.82-87 (or microcatheter) to deliver a composition disclosed herein at a controlled flow rate and in a fixed volume, via the round window membrane.
  • surgical procedures for mammals may include venting to increase AAV vector transduction rates along the length of the cochlea.
  • rodents e.g., mice, rats, hamsters, or rabbits
  • primates e.g., NHP (e.g., macaque, chimpanzees, monkeys, or apes) or humans
  • venting facilitates transduction rates of about 75-100% of IHCs throughout the cochlea.
  • venting permits IHC transduction rates of about 50-70%, about 60-80%, about 70-90%, or about 80-100% at the base of the cochlea. In some embodiments, venting permits IHC transduction rates of about 50-70%, about 60-80%, about 70-90%, or about 80-100% at the apex of the cochlea.
  • a delivery device described herein may be placed in a sterile field of an operating room and the end of a tubing may be removed from the sterile field and connected to a syringe that has been loaded with a composition disclosed herein (e.g., one or more AAV vectors) and mounted in the pump.
  • a composition disclosed herein e.g., one or more AAV vectors
  • a needle may then be passed through the middle ear under visualization (surgical microscope, endoscope, and/or distal tip camera).
  • a needle (or microneedle) may be used to puncture the RWM. The needle may be inserted until a stopper contacts the RWM.
  • the device may then be held in that position while a composition disclosed herein is delivered at a controlled flow rate to the inner ear, for a selected duration of time.
  • the flow rate (or infusion rate) may include a rate of about 30 pL/min, or from about 25 pL/min to about 35 pL/min, or from about 20 pL/min to about 40 pL/min, or from about 20 pL/min to about 70 pL/min, or from about 20 pL/min to about 90 pL/min, or from about 20 pL/min to about 100 pL/min.
  • the flow rate is about 20 pL/min, about 30 pL/min, about 40 pL/min, about 50 pL/min, about 60 pL/min, about 70 pL/min, about 80 pL/min, about 90 pL/min or aboutlOO pL/min.
  • the selected duration of time (that is, the time during which a composition disclosed herein is flowing) may be about 3 minutes, or from about 2.5 minutes to about 3.5 minutes, or from about 2 minutes to about 4 minutes, or from about 1.5 minutes to about 4.5 minutes, or from about 1 minute to about 5 minutes.
  • the total volume of a composition disclosed herein that flows to the inner ear may be about 0.09 mL, or from about 0.08 mL to about 0.10 mL, or from about 0.07 mL to about 0.11 mL. In some embodiments, the total volume of a composition disclosed herein equates to from about 40% to about 50% of the volume of the inner ear.
  • a device described herein may be configured as a single-use disposable product.
  • a device described herein may be configured as a multi use, sterilizable product, for example, with a replaceable and/or sterilizable needle sub- assembly. Single use devices may be appropriately discarded (for example, in a biohazard sharps container) after administration is complete.
  • a composition disclosed herein comprises one or a plurality of AAV vectors. In some embodiments, when more than one AAV vector is included in the composition, the AAV vectors are each different. In some embodiments, an AAV vector comprises an OTOF coding region, e.g., as described herein. In some embodiments, a composition comprises an rAAV particle comprising an AAV vector described herein. In some embodiments, the r AAV particle is encapsidated by an Anc80 capsid. In some embodiment, the Anc80 capsid comprises a polypeptide of SEQ ID NO: 109.
  • the subject or mammal is a rodent, a non-human primate, or a human. In some embodiments of any of the methods described herein, the subject or mammal is an adult, a teenager, a juvenile, a child, a toddler, an infant, or a newborn.
  • the subject or mammal is 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 2-5, 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80- 90, 90-100, 100-110, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-110, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-110, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 40-60, 40-70, 40-80, 40-90, 40-100, 50-70, 50-80, 50-90, 50-100, 60-80, 60-90, 60-100, 70-90, 70-100, 70-110, 80-100, 80-110, or 90-110 years of age. In some embodiments of any of the methods
  • the methods result in improvement in hearing (e.g., any of the metrics for determining improvement in hearing described herein) in a subject in need thereof for at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 100 days, at least 105 days, at least 110 days, at least 115 days, at least 120 days, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.
  • hearing e.g., any of the metrics for determining improvement in hearing described herein
  • the subject or mammal has or is at risk of developing non-syndromic sensorineural hearing loss.
  • the subject or mammal has been previously identified as having a mutation in an otoferlin gene.
  • the subject or mammal has any of the mutations in an otoferlin gene that are described herein or are known in the art to be associated with non-symptomatic sensorineural hearing loss.
  • the subject or mammal has been identified as being a carrier of a mutation in an otoferlin gene (e.g., via genetic testing). In some embodiments of any of the methods described herein, the subject or human has been identified as having a mutation in an otoferlin gene and has been diagnosed with non-symptomatic sensorineural hearing loss. In some embodiments of any of the methods described herein, the subject or human has been identified as having non-symptomatic sensorineural hearing loss.
  • successful treatment of non-symptomatic sensorineural hearing loss can be determined in a subject using any of the conventional functional hearing tests known in the art.
  • functional hearing tests are various types of audiometric assays (e.g., pure-tone testing, speech testing, test of the middle ear, auditory brainstem response, and otoacoustic emissions).
  • a mammalian cell is a cochlear inner hair cell.
  • a mammalian cell is a human cell (e.g., a human cochlear inner hair cell).
  • a mammalian cell is in vitro.
  • a mammalian cell is in a mammal.
  • a mammalian cell is originally obtained from a mammal and/or is cultured ex vivo. In some embodiments, a mammalian cell has previously been determined to have a defective otoferlin gene.
  • an increase in expression of an active otoferlin protein as described herein is, e.g., as compared to a control or to the level of expression of an active otoferlin protein (e.g., a full-length otoferlin protein) prior to the introduction of the vector(s).
  • the level of expression of an otoferlin protein can be detected directly (e.g., detecting otoferlin protein or detecting otoferlin mRNA).
  • Non-limiting examples of techniques that can be used to detect expression and/or activity of otoferlin directly include: real-time PCR, Western blotting, immunoprecipitation, immunohistochemistry, or immunofluorescence.
  • expression of an otoferlin protein can be detected indirectly (e.g., through functional hearing tests).
  • compositions comprising a construct as described herein.
  • a composition comprises one or more constructs as described herein.
  • a composition comprises a plurality of constructs as described herein. In some embodiments, when more than one construct is included in the composition, the constructs are each different.
  • a composition comprises an AAV vector as described herein. In some embodiments, a composition comprises one or more AAV vectors as described herein. In some embodiments, a composition comprises a plurality of AAV vectors. In some embodiments, when more than one AAV vector is included in the composition, the AAV vectors are each different. In some embodiments, an AAV vector comprises an OTOF coding region, e.g., as described herein.
  • a composition comprises one or more recombinant AAV (rAAV) particles.
  • an rAAV particle comprises a recombinant AAV vector (rAAV).
  • an rAAV particle is encapsidated by an Anc80 capsid.
  • the Anc80 capsid comprises a polypeptide of SEQ ID NO: 109.
  • a composition is or comprises a pharmaceutical composition.
  • a composition described herein is in a solution.
  • composition disclosed herein e.g., one or a plurality of AAV vectors disclosed herein, is administered as a single dose or as a plurality of doses.
  • a composition disclosed herein is administered as a single dose. In some embdiments, a composition disclosed herein is administered as a plurality of doses, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses.
  • a composition disclosed herein (e.g., a composition comprising one or a plurality of AAV vectors disclosed herein) is administered at a volume of about O.OlmL, about 0.02 mL, about 0.03 mL, about 0.04 mL, about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 1.00 mL, about 1.10 mL, about 1.20 mL, about 1.30 mL, about 1.40 mL, about 1.50 mL, about 1.60 mL, about 1.70 mL, about 1.80 mL, about 1.90 mL, or about 2.00 mL.
  • a composition disclosed herein is administered at a volume of about O.OlmL. In some embodiments, a composition disclosed herein is administered at a volume of about 0.02 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 0.03 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 0.04 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 0.05 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 0.06 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 0.07 mL.
  • a composition disclosed herein is administered at a volume of about 0.08 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 0.09 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.00 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.10 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.20 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.30 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.40 mL.
  • a composition disclosed herein is administered at a volume of about 1.50 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.60 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.70 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.80 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 1.90 mL. In some embodiments, a composition disclosed herein is administered at a volume of about 2.00 mL.
  • a composition disclosed herein (e.g., a composition comprising one or a plurality of AAV vectors disclosed herein) is administered at a volume of about 0.01 to 2.00 mL, about 0.02 to 1.90 mL, about 0.03 to 1.8 mL, about 0.04 to 1.70 mL, about 0.05 to 1.60 mL, about 0.06 to 1.50 mL, about 0.06 to 1.40 mL, about 0.07 to 1.30 mL, about 0.08 to 1.20 mL, or about 0.09 to 1.10 mL.
  • a composition disclosed herein (e.g., a composition comprising one or a plurality of AAV vectors disclosed herein) is administered at a volume of about 0.01 to 2.00 mL, about 0.02 to 2.00 mL, about 0.03 to 2.00 mL, about 0.04 to 2.00 mL, about 0.05 to 2.00 mL, about 0.06 to 2.00 mL, about 0.07 to 2.00 mL, about 0.08 to 2.00 mL, about 0.09 to 2.00 mL, about 0.01 to 1.90 mL, about 0.01 to 1.80 mL, about 0.01 to 1.70 mL, about 0.01 to 1.60 mL, about 0.01 to 1.50 mL, about 0.01 to 1.40 mL, about 0.01 to 1.30 mL, about 0.01 to 1.20 mL, about 0.01 to 1.10 mL, about 0.01 to 1.00 mL, about 0.01 to 0.09 mL.
  • compositions of the present disclosure may comprise a nucleic acid, e.g., one or a plurality of AAV vectors, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • a pharmaceutical composition may comprise one or more AAV vectors, e.g., one or more AAV constructs encapsidated by one or more AAV serotype capsids, as described herein.
  • a pharmaceutical composition may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans; mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose, or dextrans
  • mannitol proteins
  • polypeptides or amino acids such as glycine
  • antioxidants chelating agents
  • adjuvants e.g., aluminum hydroxide
  • the therapeutic compositions are formulated for intra- cochlear administration. In some embodiments, the therapeutic compositions are formulated to comprise a lipid nanoparticle. In some embodiments, the therapeutic compositions are formulated to comprise a polymeric nanoparticle. In some embodiments, the therapeutic compositions are formulated to comprise a mini-circle DNA. In some embodiments, the therapeutic compositions are formulated to comprise a CELiD DNA. In some embodiments, the therapeutic compositions are formulated to comprise a synthetic perilymph solution.
  • An exemplary synthetic perilymph solution includes 20-200mM NaCl; 1-5 mM KC1; 0.1-lOmM CaCl2; 1-lOmM glucose; and 2-50 mM HEPES, with a pH between about 6 and about 9.
  • any of the compositions described herein can further include one or more agents that promote the entry of a nucleic acid or any of the vectors described herein into a mammalian cell (e.g., a liposome or cationic lipid).
  • any of the vectors described herein can be formulated using natural and/or synthetic polymers.
  • Non-limiting examples of polymers that may be included in any of the compositions described herein can include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.), formulations from Mims Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PhaseRX polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGY® (PhaseRX, Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimers and poly (lactic-co-glycolic acid) (PLGA) polymers, RONDELTM (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, Calif.), and pH responsive co-block
  • compositions described herein can be, e.g., a pharmaceutical composition.
  • the composition includes a pharmaceutically acceptable carrier (e.g., phosphate buffered saline, saline, or bacteriostatic water).
  • a pharmaceutically acceptable carrier e.g., phosphate buffered saline, saline, or bacteriostatic water.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, injectable gels, drug-release capsules, and the like.
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into any of the compositions described herein.
  • a single dose of any of the compositions described herein can include a total sum amount of the at least two different vectors of at least 1 ng, at least 2 ng, at least 4 ng, about 6 ng, about 8 ng, at least 10 ng, at least 20 ng, at least 30 ng, at least 40 ng, at least 50 ng, at least 60 ng, at least 70 ng, at least 80 ng, at least 90 ng, at least 100 ng, at least 200 ng, at least 300 ng, at least 400 ng, at least 500 ng, at least 1 pg, at least 2 pg, at least 4 pg, at least 6 pg, at least 8 pg, at least 10 pg, at least 12 pg, at least 14 pg, at least 16 pg, at least 18 pg, at least 20 pg, at least 22 pg, at least 24 pg, at least 26 pg, at least 28 pg, at least 30
  • compositions provided herein can be, e.g., formulated to be compatible with their intended route of administration.
  • An intended route of administration is local administration (e.g., intra-cochlear administration).
  • the therapeutic compositions are formulated to include a lipid nanoparticle. In some embodiments, the therapeutic compositions are formulated to include a polymeric nanoparticle. In some embodiments, the therapeutic compositions are formulated to comprise a mini-circle DNA. In some embodiments, the therapeutic compositions are formulated to comprise a CELiD DNA. In some embodiments, the therapeutic compositions are formulated to comprise a synthetic perilymph solution.
  • An exemplary synthetic perilymph solution includes 20-200 mM NaCl; 1-5 mM KC1; 0.1- 10 mM CaCl 2 ; 1-10 mM glucose; 2-50 mM HEPES, having a pH of between about 6 and about 9.
  • kits including any of the compositions described herein.
  • a kit can include a solid composition (e.g., a lyophilized composition including the at least two different vectors described herein) and a liquid for solubilizing the lyophilized composition.
  • a kit can include a pre-loaded syringe including any of the compositions described herein.
  • a kit includes a vial comprising any of the compositions described herein (e.g., formulated as an aqueous composition, e.g., an aqueous pharmaceutical composition).
  • a kit can include instructions for performing any of the methods described herein.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.
  • Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the rAAV compositions disclosed herein may also be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • dispersion media includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
  • the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein.
  • the formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
  • Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (ML Vs).
  • MLVs generally have diameters of from 25 nm to 4 Tm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 .ANG., containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Nanocapsule formulations of the rAAV may be used.
  • Nanocapsules can generally entrap substances in a stable and reproducible way.
  • ultrafme particles sized around 0.1 Tm
  • Biodegradable polyalkyl- cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
  • compositions described herein may be administered to a subject trans arterially, subcutaneously, intradermally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • i.v. intravenous
  • nucleic acid compositions of the present disclosure are administered to a subject by intradermal or subcutaneous injection.
  • nucleic compositions of the present disclosure are administered by i.v. injection.
  • the present disclosure provides therapeutic delivery systems for treating deafness and other hearing-associated diseases, disorders and conditions.
  • therapeutic delivery systems that include i) a medical device capable of creating one or a plurality of incisions in a round window membrane of an inner ear of a human subject in need thereof, and ii) an effective dose of a therapeutic composition comprising one or a plurality of adeno-associated viral (AAV) vectors, wherein the one or the plurality of AAV vectors are capable of constituting a full-length auditory polypeptide messenger RNA in a target cell of the inner ear.
  • AAV adeno-associated viral
  • the method comprises the steps of: administering intra-cochlearly to a human subject in need thereof an effective dose of a therapeutic composition of the present disclosure, wherein the therapeutic composition is capable of being administered by using a medical device which comprises: a) a means for creating one or a plurality of incisions in a round window membrane; and b) an effective dose of a therapeutic composition.
  • the methods include the steps of: introducing into a cochlea of a human subject a first incision at a first incision point; and administering intra-cochlearly an effective dose of a therapeutic composition (e.g., any of the compositions described herein) as provided herein.
  • a therapeutic composition e.g., any of the compositions described herein
  • a therapeutic composition is administered to the subject at a first incision point.
  • a therapeutic composition is administered to a subject into or through a first incision.
  • a therapeutic composition is administered to a subject into or through a cochlea oval window membrane.
  • a therapeutic composition is administered to a subject into or through a cochlea round window membrane.
  • a composition disclosed herein can be administered to a subject with a surgical procedure.
  • administration e.g., via a surgical procedure, comprises injecting a composition disclosed herein via a delivery device as described herein into the inner ear.
  • a surgical procedure disclosed herein comprises performing a transcanal tympanotomy; performing a laser- assisted micro-stapedotomy; and injecting a composition disclosed herein via a delivery device as described herein into the inner ear.
  • a surgical procedure comprises performing a transcanal tympanotomy; performing a laser-assisted micro-stapedotomy; injecting a composition disclosed herien via a delivery device as described herein into the inner ear ; applying sealant around the round window and/or an oval window of the subject; and lowering a tympanomeatal flap of the subject to the anatomical position.
  • a surgical procedure comprises performing a transcanal tympanotomy; preparing a round window of the subject; performing a laser-assisted micro-stapedotomy; preparing both a delivery device as described herein and a composition disclosed herein for delivery to the inner ear; injecting a composition disclosed herein via the delivery device into the inner ear ; applying sealant around the round window and/or an oval window of the subject; and lowering a tympanomeatal flap of the subject to the anatomical position.
  • performing a laser-assisted micro-stapedotomy includes using a KTP otologic laser and/or a C02 otologic laser.
  • a composition comprises one or a plurality of AAV vectors. In some embodiments, when more than one AAV vector is included in the composition, the AAV vectors are each different. In some embodiments, an AAV vector comprises an OTOF coding region, e.g., as described herein. In some embodiments, a composition comprises an rAAV particle comprising an AAV vector described herein. In some embodiments, the r AAV particle is encapsidated by an Anc80 capsid. In some embodiment, the Anc80 capsid comprises a polypeptide of SEQ ID NO: 109.
  • a therapeutic composition is administered using a medical device capable of creating a plurality of incisions in a round window membrane.
  • a medical device includes a plurality of micro-needles.
  • a medical device includes a plurality of micro-needles including a generally circular first aspect, wherein each micro-needle has a diameter of at least about 10 microns.
  • a medical device includes a base and/or a reservoir capable of holding a therapeutic composition.
  • a medical device includes a plurality of hollow micro-needles individually including a lumen capable of transferring a therapeutic composition.
  • a medical device includes a means for generating at least a partial vacuum.
  • a composition disclosed herein is administered using a device and/or system specifically designed for intracochlear route of administration.
  • design elements of a device described herein may include: maintenance of sterility of injected fluid; minimization of air bubbles introduced to the inner ear; ability to precisely deliver small volumes at a controlled rate; delivery through the external auditory canal by the surgeon; minimization of damage to the round window membrane (RWM), or to inner ear, e.g., cochlear structures beyond the RWM; and/or minimization of injected fluid leaking back out through the RWM.
  • RWM round window membrane
  • the devices, systems, and methods provided herein also describe the potential for delivering a composition safely and efficiently into the inner ear, in order to treat conditions and disorders that would benefit from delivery of a composition disclosed herein to the inner ear, including, but not limited to, hearing disorders, e.g., as described herein.
  • a composition disclosed herein is dispersed throughout the cochlea with minimal dilution at the site of action.
  • the development of the described devices allows the surgical administration procedure to be performed through the external auditory canal in humans.
  • the described devices can be removed from the ear following infusion of an amount of fluid into the perilymph of the cochlea.
  • the device may be advanced through the external auditory canal, either under surgical microscopic control or along with an endoscope.
  • FIGs. 81-84 An exemplary device for use in any of the methods disclosed herein is described in FIGs. 81-84.
  • Fig. 81 illustrates an exemplary device 10 for delivering fluid to an inner ear.
  • Device 10 includes a knurled handle 12, and a distal handle adhesive 14 (for example, an epoxy such as loctite 4014) that couples to a telescoping hypotube needle support 24.
  • the knurled handle 12 (or handle portion) may include kurling features and/or grooves to enhance the grip.
  • the knurled handle 12 may be from about 5 mm to about 15 mm thick or from about 5 mm to about 12 mm thick, or from about 6 mm to about 10 mm thick, or from about 6 mm to about 9 mm thick, or from about 7 mm to about 8 mm thick.
  • the knurled handle 12 (or handle portion) may be hollow such that fluid may pass through the device 10 during use.
  • the device 10 may also include a proximal handle adhesive 16 at a proximal end 18 of the knurled handle 12, a needle sub-assembly 26 (shown in Fig. 82) with stopper 28 (shown in Fig.
  • Strain relief feature 22 may be composed of a Santoprene material, a Pebax material, a polyurethane material, a silicone material, a nylon material, and/or a thermoplastic elastomer.
  • the telescoping hypotube needle support 24 surrounds and supports a bent needle 38 (shown in Fig. 82) disposed therewithin.
  • the stopper 28 may be composed of a thermoplastic material or plastic polymer (such as a UV-cured polymer), as well as other suitable materials, and may be used to prevent the bent needle 38 from being inserted too far into the ear canal (for example, to prevent insertion of bent needle 38 into the lateral wall or other inner ear structure).
  • Device 10 also may include a tapered portion 23 disposed between the knurled handle 12 and the distal handle adhesive 14 that is coupled to the telescoping hypotube needle support 24.
  • the knurled handle 12 (or handle portion) may include the tapered portion 23 at the distal end of the handle portion 12.
  • Device 10 may also include tubing 36 fluidly connected to the proximal end 16 the device 10 and acts as a fluid inlet line connecting the device to upstream components (for example, a pump, a syringe, and/or upstream components which, in some emboidments, may be coupled to a control system and/or power supply (not shown)).
  • upstream components for example, a pump, a syringe, and/or upstream components which, in some emboidments, may be coupled to a control system and/or power supply (not shown)).
  • the bent needle 38 (shown in Fig. 82) extends from the distal end 20, through the telescoping hypotube needle support 24, threough the tapered portion 23, through the knurled handle 12, and through the strain relief feature 22 and fluidly connects directly to the tubing 36.
  • the bent needle 38 fluidly connects with the hollow interior of the knurled handle (for example, via the telescoping hypotube needle support 24) which in turn fluidly connects at a proximal end 16 with tubing 36.
  • the contact area for example, between overlapping nested hyotubes 42
  • the tolerances, and/or sealants between interfacing components must be sufficent to prevent therapeutic fluid from leaking out of the device 10 (which operates at a relatively low pressure (for example, from about 1 Pascal to about 50 Pa, or from about 2 Pa to about 20 Pa, or from about 3 Pa to about 10 Pa)).
  • Fig. 82 illustrates a sideview of the bent needle sub-assembly 26, according to aspects of the present disclosed embodiments.
  • Bent needle sub-assembly 26 includes a needle 38 that has a bent portion 32.
  • Bent needle sub-assembly 26 may also include a stopper 28 coupled to the bent portion 32.
  • the bent portion 32 includes an angled tip 34 at the distal end 20 of the device 10 for piercing a membrane of the ear (for example, the RWM).
  • the needle 38, bent portion 32, and angled top 34 are hollow such that fluid may flow therethrough.
  • the angle 46 (as shown in Fig. 84) of the bent portion 32 may vary.
  • a stopper 28 geometry may be cyclidrical, disk-shaped, annulus-shaped, dome-shaped, and/or other suitable shapes. Stopper 28 may be molded into place onto bent portion 32. For example, stopper 28 may be positioned concentrically around the bent portion 32 using adhesives or compression fitting. Examples of adhesives include an UV cure adhesive (such as Dymax 203 A-CTH-F-T), elastomer adhesives, thermoset adhesives (such as epoxy or polyurthethane), or emulsion adhesives (such as polyvinyl acetate). Stopper 28 fits concentrically around the bent portion 32 such that angled tip 34 is inserted into the ear at a desired insertion depth.
  • the bent needle 38 may be formed from a straight needle using incremental forming, as well as other suitable techniques.
  • Fig. 83 illustrates a perspective view of exemplary device 10 for delivering fluid to an inner ear.
  • Tubing 36 may be from about 1300 mm in length (dimension 11 in Fig. 83) to about 1600 mm, or from about 1400 mm to about 1500 mm, or from about 1430 mm to about 1450 mm.
  • Strain release feature 22 may be from about 25 mm to about 30 mm in length (dimension 15 in Fig. 83), or from about 20 mm to about 35 mm in length.
  • Handle 12 may be about 155.4 mm in length (dimension 13 in Fig. 83), or from about 150 mm to about 160 mm, or from about 140 mm to about 170 mm.
  • the telescoping hypotube needle support 24 may have two or more nested hypotubes, for example three nested hypotubes 42A, 42B, and 42C, or four nested hypotubes 42A, 42B, 42C, and 42D .
  • the total length of hypotubes 42 A, 42B, 42C and tip assembly 26 may be from about 25 mm to about 45 mm, or from about 30 mm to about 40 mm, or about 35 mm.
  • telescoping hypotube needle support 24 may have a length of about 36 mm, or from about 25 mm to about 45 mm, or form about 30 mm to about 40 mm.
  • the three nested hypotubes 42A, 42B, and 42C each may have a length of 3.5 mm, 8.0 mm, and 19.8 mm, respectively, plus or minus about 20%.
  • the inner-most nested hypotube (or most narrow portion) of the telescoping hypotube needle support 24 may be concentrically disposed around needle 38 .
  • Fig. 84 illustrates a perspective view of bent needle sub-assembly 26 coupled to the distal end 20 of device 10, according to aspects of the present disclosed embodiments.
  • bent needle sub-assembly 26 may include a needle 38 coupled to a bent portion 32.
  • the bent needle 38 may be a single needle (for example, a straight needle that is then bent such that it includes the desired angle 46).
  • Needle 38 may be a 33-gauge needle, or may include a gauge from about 32 to about 34, or from about 31 to 35. At finer gauges, care must be taken to ensure tubing 36 is not kinked or damaged. Needle 38 may be attached to handle 12 for safe and accurate placement of needle 38 into the inner ear.
  • bent needle sub-assembly 26 may also include a stopper 28 disposed around bent portion 32.
  • bent portion 32 may include an angled tip 34 for piercing a membrane of the ear (for example, the RWM).
  • Stopper 28 may have a height 48 of about 0.5 mm, or from about 0.4 mm to about 0.6 mm, or from about 0.3 mm to about 0.7 mm.
  • Bent portion 32 may have a length 52 of about 1.45 mm, or from about 1.35 mm to about 1.55 mm, or from about 1.2 mm to about 1.7 mm.
  • the bent portion 32 may have a length greater than 2.0 mm such that the distance between the distal end of the stopper 28 and the distal end of the angled tip 34 is from about 0.5 mm to about 1.7 mm, or from about 0.6 mm to about 1.5 mm, or from about 0.7 mm to about 1.3 mm, or from about 0.8 mm to about 1.2 mm.
  • Fig. 84 shows that stopper 28 may have a geometry that is cyclidrical, disk-shaped, and/or dome-shaped. A person of ordinary skill will appreciate that other geometries could be used.
  • Example 1 Characterization of human Otoferlin Gene, Homologs, Orthologs.
  • the otoferlin gene and the corresponding mRNA are provided below.
  • Recombinant AAV is generated by transfection with an adenovirus-free method as used by Xiao et al. J Virol 1999, 73(5):3994-4003.
  • the cis plasmids with AAV ITRs, the trans plasmid with AAV Rep and Cap genes, and a helper plasmid with an essential region from an adenovirus genome are co-transfected in 293 cells in a ratio of 1 : 1 :2.
  • the AAV vectors used here express human otoferlin or mouse otoferlin under multiple dual vector strategies using the constructs described below.
  • AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rhlO, rh39, rh43, and Anc80 are each prepared to encapsulate three sets of otoferlin constructs to test (i) a concatemerization-trans-splicing strategy, (ii) a hybrid intronic-homologous recombination-trans-splicing strategy, and (iii) an exonic homologous recombination strategy, as summarized by Pryadkina et al. Meth Clin Devel 2015, 2:15009.
  • Oligo-dT and random-primed cDNA libraries are constructed from poly(A)+ mRNA of human total fetus, adult brain, heart, kidney, and murine fetal heart as described by Yasunaga et al. Am J Genet 2000, 67:591-600. RACE-PCR experiments are performed on these libraries using linker primers and a series of primers selected from the otoferlin cDNA sequence. The PCR products are directly cloned into pGEM-T Easy vector and sequenced.
  • a reverse primer (5’-TTCACCTGGGCCCGCA-GCATCCT-3’ (SEQ ID NO: 29)) is designed from the sequence encoding aa 63-70 of the initially reported short form of otoferlin (Yasunaga et al., 1999) (GenBank 107403).
  • Total RNAs are extracted from mouse cochlea using the methods described in Strenzke et al., EMBO ./. 35(23):2519-2535, 2016. RT-PCR experiments are performed in various murine and human RNA sources, according to the GeneAmp RNA PCR kit.
  • Two primer pairs are used to reconstitute the murine cDNAs derived from the brain and the cochlea, one from the exon 1 5’-UTR (5’-AGGCGTGTGAGCCACACTCCACCA-3’ (SEQ ID NO: 30)) and exon 22 (5’-CATAACCTCAGCTTGTCCCGAACA-3’ (SEQ ID NO: 31)), and the other from the exon 18-19 junction (5’-
  • the 5’ cassette includes a synthetic hair cell-oriented promoter, a chimeric intron (b-globin), a consensus Kozak sequence, the exons 1 to 26 of otoferlin and the half intron 26 of otoferlin (representing 3,836 bp, or the 3,494 bp of otoferlin cDNA corresponding to exons 1 through 26, plus the first 342 bp of intron 26), and the 3’ cassette includes the second half of intron 26 (342 bp), exons 27 to 48 (3,843 bp) of otoferlin, and a polyadenylation signal sequence.
  • the 5’ cassette includes a synthetic hair cell-oriented promoter, a chimeric intron (b-globin), a consensus Kozak sequence, the exons 1 to 26 of otoferlin and the half intron 26 of otoferlin (representing 3,836 bp, or the 3,494 bp of otoferl
  • the a hair cell-oriented promoter is not required for expression of an otoferlin protein in an auditory inner hair cell.
  • the cassettes from the concatemerization-splicing strategy described above are modified such that the full length intron 26 of otoferlin is added in the place of the half intron 26 in both plasmids.
  • the two cassettes are composed such that the 5’ cassette includes a hair cell-oriented promoter, a chimeric intron, a consensus Kozak sequence and the exons 1 to 28 (the first 3,776 bp of the otoferlin cDNA), and the 3’ cassette includes the exons 23 to 48 (the final 4,446 bp of the otoferlin cDNA) and a polyadenylation signal sequence.
  • the region of homology between the two cassettes is 885 bp.
  • Recombinant AAV-1 is produced using a triple transfection protocol and purified by two sequential cesium chloride (CsCl) density gradients, as described by Pryadkina et al. Mol Ther 2015, 2: 15009. At the end of second centrifugation, 11 fractions of 500 pi are recovered from the CsCl Density Gradient tube and purified through dialysis in 1 c PBS. The fractions are analyzed by dot blot to determine those containing rAAV genomes. The viral genome number (vg) of each preparation is determined by quantitative real-time PCR-based titration method using primers and probe corresponding to the ITR region of the AAV vector genome (Bartoli et al. Gene Ther 2006, 13:20-28).
  • CsCl cesium chloride
  • AAV produced at a titer of lel4 vg / mL is prepared at dilutions of 3.2el3,
  • Artificial perilymph is prepared by combining the following reagents, in mM: NaCl, 120; KC1, 3.5; CaCl2, 1.5; glucose, 5.5; HEPES, 20.
  • the artificial perilymph is titrated with NaOH to adjust its pH to 7.5
  • human retinal epithelial cells and neonate mouse cochlear explants are incubated with AAV-OTOF at titers of 3.2el3, 1.0el3, 3.2el2, 1.0el2 viral genome-containing particles (vg / mL) and assayed for levels of otoferlin DNA, mRNA and protein as described previously (Duncker et al., 2013 JNeurosci 33(22):9508-9519.
  • Antibodies against mouse otoferlin are obtained from Abeam and used as described by Engel et al., 2006 Neurosci 143:837-849.
  • the AAV-OTOF formulation is delivered to the cochlea using a specialized microcatheter designed for consistent and safe penetration of the RWM.
  • the microcatheter is shaped such that the surgeon performing the delivery procedure can enter the middle ear cavity via the external auditory canal and contact the end of the microcatheter with the RWM.
  • the distal end of the microcatheter is comprised of at least one microneedle with diameter of between 10 and 1,000 microns, which produce perforations in the RWM that are sufficient to allow AAV-OTOF to enter the cochlear perilymph of the scala tympani at a rate of approximately 1 pL/min, but heal without surgical repair.
  • the remaining portion of the microcatheter, proximal to the microneedle(s), is loaded with the AAV-OTOF/artificial perilymph formulation at a titer of approximately lel3 vg/mL.
  • the proximal end of the microcatheter is connected to a micromanipulator that allows for precise, low volume infusions of approximately 1 TL / min.
  • Otoferlin rescue with cochlear delivery of AAV-OTOF is assessed in three OTOF knockout mouse models (mouse models as described in Longo-Guess et al. Hearing Res 2007, 234(l-2):21-28; Roux et al. Cell 2006, 127:277-289; and Reisinger et al., J. Neurosci. 31(13):4886-4895, 2011).
  • Rescue experiments are tested in neonate (PI), juvenile (P6 or P12) and adult (P42) mice, in order to evaluate the postnatal treatment window relative to stage of cochlear development.
  • ABR auditory brainstem response
  • DPOAE distortion product optoacoustic emissions
  • IHC inner hair cell
  • OHC outer hair cell
  • All animals are expected to display the characteristic audiometric profile of otoferlin dysfunction - i.e., abnormal ABRs across tested sound frequencies but normal DPOAEs, indicative of dysfunctional IHC signal transduction and normal OHC function (Yasunaga et al. 2000, Am J Hum Genet 67:591-600).
  • ABR and DPOAE measurements are taken again bilaterally in the juvenile and adult animals 1, 5 and 10 days following the surgical procedure.
  • cochlear tissue samples are collected from the same basal, middle and apical regions as described above, and assayed for otoferlin mRNA transcript as described previously (Duncker et al. 2013, JNeurosci 33(22):9508-9519, Heidrych et al. 2008, Hum Mol Genet 17:3814-3821, Heidrych et al., 2009, Hum Mol Genet 18:2779-2790).
  • Example 9 Animal Model 1A: Surgical Method in Aged Mice
  • AAV-OTOF prepared in artificial perilymph is administered to the scala tympani in mice as described by Shu et al. 2016 (Shu Yilai, Tao Yong, Wang Zhengmin, Tang Yong, Li Huawei, Dai Pu, Gao Guanping, and Chen Zheng-Yi. Human Gene Therapy. June 2016, ahead of print doi: 10.1089/hum.2016.053).
  • Six-week-old male mice are anesthetized using an intraperitoneal injection of xylazine (20 mg/kg) and ketamine (100 mg/kg). Body temperature is maintained at 37 °C using an electric heating pad. An incision is made from the right post-auricular region and the tympanic bulla is exposed.
  • the bulla is perforated with a surgical needle and the small hole is expanded to provide access to the cochlea.
  • the bone of the cochlear lateral wall of the scala tympani is thinned with a dental drill so that the membranous lateral wall is left intact.
  • a Nanoliter Microinjection System in conjunction with glass micropipette is used to deliver a total of approximately 300 nL of AAV-OTOF in artificial perilymph to the scala tympani at a rate of 2 nL/second.
  • the glass micropipette is left in place for 5 minutes post-injection.
  • the opening in the tympanic bulla is sealed with dental cement, and the muscle and skin are sutured.
  • the mice are allowed to awaken from anesthesia and their pain is controlled with 0.15 mg/kg buprenorphine hydrochloride for 3 days.
  • AAV-OTOF prepared in artificial perilymph is administered to guinea pigs to assess distribution and toxicity following intracochlear delivery with a reciprocating micropump as described by Tandon et al. Lab Chip 2015 (DOI: 10.1039/c51c01396h).
  • Lidocaine with epinephrine is given subcutaneously at the incision site as a topical anesthetic.
  • a 5 mm diameter hole is made in the bulla and a cochleostomy is created approximately 0.5 mm distal to the round window membrane.
  • the cannula of the micropump (described below) is inserted into the cochleostomy, threaded into the cochlea 3 mm apically, and glued to the bulla with a common cyanoacrylate glue.
  • CAP compound action potential
  • a perfluoroalkoxy-alkane-insulated silver wire electrode (203 pm uncoated diameter) is inserted near the round window niche and glued to the bulla.
  • DPOAEs distortion product otoacoustic emissions
  • CAPs CAPs
  • Micropump description AAV-OTOF at a maximum titer of lel4 vg/mL is administered to the guinea pig using a micropump as described by Tandon et al. Lab Chip 2015 (DOI: 10.1039/c51c01396h).
  • the micropump system has 4 selectable ports. These ports are connected to: (i) a large fluidic capacitor used for artificial perilymph storage; (ii) an outlet that connects to the cochlea; (iii) the outlet from an integrated AAV-OTOF reservoir; (iv) the inlet to the integrated AAV-OTOF reservoir.
  • Each port is fluidically connected to a central pump chamber, and each is individually addressed with a valve.
  • the sequence of events for reciprocating AAV-OTOF delivery is as follows: (i) an internal AAV-OTOF-refresh loop is run, transferring AAV-OTOF from the AAV-OTOF reservoir into the main infuse-withdraw line; (ii) AAV-OTOF is infused into the cochlea and some artificial perilymph is drained from the artificial perilymph storage capacitor; (iii) the first two steps can be repeated several times for additional doses; (iv) after the AAV-OTOF has been allowed to diffuse for some time, a volume of perilymph is withdrawn from the cochlea that is equal to the volume infused in steps (i)-(iii), refilling the artificial perilymph storage capacitor. This process results in net delivery of drug with zero net fluid volume added to the cochlea.
  • the fluidic capacitors in the micropump are cylindrical chambers whose ceilings are a thin (25.4 pm), flexible, polyimide membrane.
  • the pump chamber has a diameter of 3.5 mm
  • the fluidic storage capacitor has a diameter of 14 mm
  • all of the remaining capacitors have diameters of 4 mm.
  • the same membrane is deflected to block flow at each of the valves.
  • the valve chambers have diameters of 3.1 mm.
  • the serpentine channel that comprises the drug reservoir has a square cross section of width 762 pm and a length of 410 mm for a total volume of 238 pL. All of the other microchannels in the pump have a width of 400 pm and a height of 254 pm.
  • the micropump is loaded with AAV-OTOF and artificial perilymph, and the cannula inserted into a cochleostomy made in the region of the cochlea between the locations with characteristic frequency sensitivity of 24 and 32 kHz, and threaded apically 3 mm, terminating in the 12-16 kHz region.
  • Baseline DPOAE and CAP hearing tests are performed prior to the start of AAV-OTOF/artificial perilymph infusion.
  • the pump is then activated and approximately 1 pL of artificial perilymph is infused every 5 min until a total of approximately 10 pL of artificial perilymph is delivered to the cochlea. After a 20 min wait time, approximately 10 pL of perilymph is withdrawn from the cochlea.
  • AAV-OTOF delivery is then initiated at a rate of approximately 1 pL every 5 min until a total of approximately 10 pL of fluid is delivered.
  • Extent of AAV transduction and OTOF expression along the organ of Corti is assessed via immunostaining with anti-OTOF antibodies.
  • Antibodies against markers for hair cells (Myo7a) and supporting cells (Sox2) are used to quantify IHCs, OHCs, supporting cells and stereocilia morphology.
  • Annexin V staining is used to assess evidence of apoptosis in cells along the cochlear sensory epithelium.
  • AAV-OTOF prepared in artificial perilymph is administered to juvenile sheep to assess distribution and toxicity following delivery to the cochlea via trans-RWM infusion.
  • IHC inner hair cell
  • OOC outer hair cell
  • ABR and DPOAE measurements are taken again bilaterally 1, 5 and 10 days following the surgical procedure. At 6 months post-procedure, additional bilateral ABR and DPOAE measurements are taken from all animals, and the animals are subsequently sacrificed and their cochleae removed.
  • cochlear tissue samples are collected from the same basal, middle and apical regions as described above, and assayed for otoferlin mRNA transcript as described previously (Duncker et al. 2013, JNeurosci 33(22):9508-9519, Heidrych et al. 2008, Hum Mol Genet 17:3814-3821, Heidrych et al., 2009, Hum Mol Genet 18:2779- 2790).
  • the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene plasmid #42230) is digested with Bsbl, dephosphorylated using Antartic Phosphatase, and the linearized vector is gel purified.
  • pX330-cas9-OTOF bicistronic vector
  • a pair of oligos for targeting otoferlin exon 1 is annealed, phosphorylated and ligated to a linearized vector (Cong et al. 2013 Science 339(6121): 819-23).
  • the Al 5 astroglial sheep cell line (Vilette et al., 2000 In Vitro Cell Dev Biol Anim 36(l):45-9) is maintained in DMEM in 10% Fetal Bovine Serum, 2mM glutamine, 1% sodium pyruvate and 1% penicillin/streptomycin.
  • Cells are transfected in 24-well plates with 2 pg of pX330-cas9-OTOF co-expressing Cas9 and sgRNA against otoferlin using lipofectamine LTX reagent.
  • genomic DNA from transfected cells is extracted and quantified using a NanoDrop2000 spectrophotometer, measuring A260/A280 andA260/A230 ratios to account for sample purity
  • Gene mutation activity of sgRNA sequence at the target locus of OTOF exon 1 is quantified using the T7EI mismatch detection assay.
  • DNA sequence of interest is PCR- amplified with a high-fidelity polymerase (Herculase II fusion polymerase) using specific primers.
  • the resultant PCR product is then denatured and slowly re-annealed (95 °C, 2 min; 95°C to 85°C, -2°C/sec; 85°C to 25°C, -l°C/sec) to produce homoduplex/heteroduplex mix. This is then digested by 5U of T7EI restriction enzyme at 37°C for 30 minutes. Digestion products are separated by 2% agarose gel electrophoresis.
  • T7 promoter is added to sgRNA template by PCR amplification of pX330-cas9-OTOF plasmid.
  • the PCR product is purified using NucleoSpin Gel and PCR Clean-up. It is used as the template for in vitro transcription using MEGAshortscript T7 kit according to the manufacturer’s manual. Following completion of transcription, DNase I treatment is performed.
  • the Cas9 mRNA is transcribed using Pmel-digested Cas9 expression JDS246 plasmid (Addgene plasmid # 43861) and the mMESSAGE mMACHINE T7 ULTRA Transcription Kit according to the manufacturer’s manual. Following completion of transcription, the poly(A) tailing reaction and DNase I treatment are performed. Both the Cas9 mRNA and the sgRNAs are purified using MEGAclear kit and eluted in elution buffer.
  • the embryos are produced by in vitro fertilization according to routine procedure as described previously (Crispo et al. 2014 Transgenic Res, 24(1):31-41). Briefly, ovaries from slaughterhouse are transported to the laboratory and cumulus oocyte complexes (COCs) are aspirated in recovery medium. The selected COCs are placed in maturation medium for 24 h in 5% C02 in humidified air atmosphere at 39°C. Then, expanded COCs are inseminated in 100 pi drops with 1 x 106 dose of frozen-thawed semen selected by ascendant migration on a swim up method. Fertilization is carried out in 5% C02 with humidified atmosphere at 39°C for 22 h.
  • injection buffer lOmM Tris pH 7.5, 0. ImM EDTA
  • Buffer group is injected with the same procedure but with buffer alone.
  • injected and non-injected embryos are transferred to culture medium under mineral oil, in 5% C02, 5% 02 and 90% N2 in humidified atmosphere at 39°C.
  • Cleavage rate on Day 2 (cleaved zygotes per total oocytes) and development rate on Day 6 (morulae and blastocysts per total oocytes) are recorded for all experimental groups.
  • DNA from 20 CRISPR group embryos are analyzed by Sanger sequencing to detect the mutation at the OTOF gene level.
  • blastocysts produced by CRISPR/Cas9 zygote microinjection are transferred to 29 recipient females. Only early blastocysts, blastocysts and expanded blastocysts classified as excellent or good (i.e. Grade 1 as defined in Stringfellow et al. 2010, Manual of the International Embryo Transfer Society) are transferred on Day 6 after fertilization. Embryo transfer is performed by minimally invasive surgery assisted by laparoscopy to place the embryos into the cranial side of the ipsilateral uterine horn to the corpus luteum.
  • Recipient ewes are previously synchronized to be on Day 6 of the estrous cycle using a standard protocol to control ovulation described previously, as described by Menchaca et al. 2004, Reprod Fertil Dev. 16(4):403-413.
  • Pregnancy diagnosis and fetal development are performed on Day 30 and 105, respectively, by using B-mode ultrasonography equipped with a 5 and 3.5 MHz probe.
  • Day 0 of the experiment is defined as the moment of embryo fertilization.
  • Several parameters are measured to study the development of fetuses at Day 105 of gestation: thoracic diameter, biparietal diameter, occipitonasal length and heart rate.
  • body weight, thoracic perimeter, biparietal diameter, crown-rump and occipitonasal length, height at withers, height at hips, width at hips and width at chest were recorded.
  • Body weight and morphometric variables are determined at birth, and 15, 30 and 60 days later.
  • Samples from skin and limb muscle of the lambs are taken seven days after birth and T7EI assay, western blot test and histology examinations are performed in order to identify and characterize KO founders and off-target sites.
  • Total DNA is isolated from skin biopsies for all animals and from muscle for some animals. Samples are analyzed using capillary electrophoresis. Genotyping of OTOF exon 1 is performed by direct sequencing of PCR amplicons and in muscle biopsies by additional sequencing of isolated bacterial clones with individual amplicon sequences.
  • Western blotting is performed to determine the presence of myostatin in the muscle fiber. Equal amounts of total proteins are run on 12% (v/v) gel electrophoresis and electrophoretically transferred to a PVDF membrane. Monoclonal mouse anti- otoferlin antibody is used in the western blotting. The washed membranes are incubated with 1:50000 dilution of secondary antibody linked to horseradish peroxidase (HPR). HPR activity is detected using western blot chemiluminescence.
  • HPR horseradish peroxidase
  • AAV-OTOF prepared in artificial perilymph is administered to OTOF knockout transgenic sheep to assess the ability to restore normal hearing function following delivery to the cochlea via trans-RWM infusion.
  • IHC inner hair cell
  • OOC outer hair cell
  • ABR and DPOAE measurements are taken again bilaterally 1, 5 and 10 days following the surgical procedure. At 6 months post procedure, additional bilateral ABR and DPOAE measurements are taken from all animals, and the animals are subsequently sacrificed and their cochleae removed.
  • cochlear tissue samples are collected from the same basal, middle and apical regions as described above, and assayed for otoferlin mRNA transcript as described previously (Duncker et al. 2013, J Neurosci 33(22):9508-9519, Heidrych et al. 2008, Hum Mol Genet 17:3814-3821, Heidrych et al., 2009, Hum Mol Genet 18:2779- 2790).
  • Example 13 Human Clinical Example (Pediatric Treatment)
  • the subject is put under general anesthesia.
  • the surgeon approaches the tympanic membrane from external auditory canal, makes a small incision at the inferior edge of the external auditory canal where it meets the tympani membrane, and lifts the tympanic membrane as a flap to expose the middle ear space.
  • a surgical laser is used to make a small opening (approximately 2 mm) in the stapes footplate.
  • the surgeon then penetrates the round window membrane with a microcatheter loaded with a solution of AAV-OTOF prepared in artificial perilymph at a titer of lel3 vg/mL.
  • the microcatheter is connected to a micromanipulator that infuses approximately 20 uL of the AAV-OTOF solution at a rate of approximately 1 uL / min.
  • the surgeon withdraws the microcatheter and patches the holes in the stapes foot plate and RWM with a gel foam patch. The procedure concludes with replacement of the tympanic membrane flap.
  • Example 14 Non-Invasive Prenatal Testing of Maternal Blood to Detect OTOF Mutation
  • Maternal blood samples (20-40 mL) are collected into Cell-free DNA tubes. At least 7 mL of plasma is isolated from each sample via a double centrifugation protocol of 2,000 g for 20 minutes, followed by 3,220 g for 30 minutes, with supernatant transfer following the first spin.
  • cfDNA is isolated from 7-20 mL plasma using a QIAGEN QIAmp Circulating Nuclei Acid kit and eluted in 45 pL TE buffer. Pure maternal genomic DNA is isolated from the buffy coat obtained following the first centrifugation.
  • Example 15 Alternative Examples (mRNA, Single Viral Vector, Non-Viral Vectors)
  • the C2D and C2E domains bind Ca 2+ as well as phosphatidyl serine (PS) in a Ca 2+ -dependent manner.
  • a cDNA is produced that encodes a truncated form of otoferlin lacking the C2A, C2D and C2E domains. This cDNA is suitable for packaging in an AAV vector.
  • the truncated otoferlin construct (OTOFAC2ADE) is derived and cloned from an original wildtype otoferlin plasmid encoding the full OTOF gene, as described by Padmanarayana et al. 2014 Biochem 53:5023-5033. Deletion of the coding region of the C2 domains is performed by PCR mutagenesis using domain-spanning oligonucleotides and a QuikChange site-directed mutagenesis kit applying the double mutagenic primer approach.
  • the PCR is performed as follows: 95°C for 3 minutes; 18 cycles at 95 °C for 15 seconds, 65 °C for 1 minute, and 68 °C for 12 minutes; and 68 °C for 7 minutes.
  • the PCR product is digested with Dpnl, cloned into the DSC-B vector, and transformed into DH5alpha or XL 10-Gold bacterial cells. Plasmid DNA is isolated by mini preparations and subsequently sequenced.
  • a plasmid containing a CB A promoter, a chimeric intron (b-globin), a consensus Kozak sequence, the OTOFAC2ADE cDNA and a polyadenylation signal sequence is used for the AAV construct.
  • Recombinant AAV is generated by transfection with an adenovirus-free method as used by Xiao et al. J Virol 1999, 73(5):3994-4003.
  • the cis plasmids with AAV ITRs, the trans plasmid with AAV Rep and Cap genes, and a helper plasmid with an essential region from an adenovirus genome are co-transfected in 293 cells in a ratio of 1:1:2.
  • AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rhlO, rh39, rh43, and Anc80 are each prepared to encapsulate the OTOFAC2ADE cDNA construct.
  • the otoferlin gene is prepare for non-viral gene transfer as described by Li et al. 2013, PLoS ONE 8(8):e69879. First, Spodoptera frugiperda Sf9 cells are grown in suspension in serum-free media. The blasticidin-S deaminase (bs) gene is PCR-amplified from pIB/V5-His/CAT using the following primer pair:
  • the bsd r clones are expanded in insect cell culture medium supplemented with 10% FBS and blasticidin-S HC1 (10 pg/mL) for 2 to 3 additional passages, then returned to serum-free medium with 10 pg/mL blasticidin-S HC1. After an additional 12 passages, blasticidin-S HC1 is omitted from the medium and the cell lines are expanded for analysis.
  • Clonal SfMTR-OTOF cells with the highest levels of OTOF expression are expanded for CELiD-OTOF DNA preparation.
  • Extrachromosomal DNA is extracted from the Bac-Rep-infected, SfMTR-GFP cells using a commercially available plasmid isolation kit. CELiD production is monitored by agarose gel electrophoresis and ethidium bromide staining of extrachromosomal DNA.
  • CELiD DNA is produced in parental Sf9 cells by co-infection with two separate baculovirus expression vectors (BEV): Bac-Rep and a second BEV bearing an ITR-flanked transgene, such as Bac- OTOF. Infected Sf9 cells are harvested once the mean cell diameter increases by 4-5 pm and the percent viability decreases to 80-90%. CELiD DNA is isolated using a commercially available plasmid purification kit.
  • BEV baculovirus expression vectors
  • Clonal Sf9/ITR-OTOF cells are inoculated with various amounts of Bac-Rep stock. Cells are periodically harvested and extrachromosomal DNA is recovered using a commercially available DNA isolation kit. Extracted DNA is examined by either agarose gel electrophoresis or by PCR with OTOF-specific primer pairs for quantitative determination of CELiD DNA amounts. For western blotting, cell proteins are fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes are incubated in blocking buffer (BB) composed of 5% non- fat dry milk (w:v) in phosphate-buffered saline plus 0.05% Tween-20 (PBST) for 1 hr at ambient temperature with orbital agitation.
  • BB blocking buffer
  • PBST phosphate-buffered saline
  • WB wash buffer
  • BB wash buffer
  • Non-conjugated mAbs are incubated with secondary antibody solution (goat, anti-mouse horseradish peroxidase (HRP)-conjugate for 1 hr, and then washed with WB as above. HRP activity is detected by enhanced chemiluminescence (ECL).
  • secondary antibody solution goat, anti-mouse horseradish peroxidase (HRP)-conjugate for 1 hr, and then washed with WB as above.
  • HRP activity is detected by enhanced chemiluminescence (ECL).
  • Otoferlin cDNA or mRNA is encapsulated in poly(lactic-co-glycolic acid) nanoparticles by the double-emulsion solvent evaporation method described previously (O’Donnell and McGinity 1997 Adv Drug Delivery Rev 28(l):25-42), and in lipid nanoparticles (Pezzoli et al. 2013 Methods Mol Biol 1025:269-279). Briefly, solid lipid nanoparticles can be generated from a microemulsion using Precirol ATO-5 and stearylamine as the cationic lipid.
  • Precirol ATO-5 500 mg is heated to 10°C above its melting point, and 10 mL of a hot aqueous solution of poloxamer and stearylamine in different proportions (1/1.25; 1/1.87; 1/3.12; 1/4.37 and 1/5) is added. The sample is stirred for 30 minutes at 14,000 rpm.
  • the nanoparticles are generated by dispersing the hot microemulsion in cold water (2-5 °C) in an emulsion: water ratio of 1 :5.
  • the resultant suspension is centrifuged for three times at 3,000 rpm for 20 minutes at a temperature of 20°C, reconstituting the precipitate after centrifugation.
  • Cationic solid lipid nanoparticles are lyophilized by being added an aqueous solution of cryoprotectant (5% mannitol) in a 1 :2 (SLN: mannitol) ratio.
  • the freezing temperature is set at -40°C in the lyophilizer and samples are kept at this temperature for 2 hours.
  • Lyophilization temperature is then set to 25 °C at a pressure of 0.2-0.4 mBa for 48 hours.
  • a solution of the OTOF cDNA plasmid is prepared to a concentration of 2pg/pL.
  • a 25 pL aliquot of the plasmid DNA solution is then added to different volumes of the cationic SLN suspension to obtain ratios of between 15:1 and 1:1 (SLNOTOF) by stirring.
  • mRNA or modified mRNA, or “mmRNA” for use in accordance with the disclosure may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription (IVT), or enzymatic or chemical cleavage of a longer precursor, etc.
  • IVTT in vitro transcription
  • Methods of synthesizing RNAs are known in the art (see, e.g., Gait,
  • the process of design and synthesis of the primary constructs of the disclosure generally includes the steps of gene construction, mRNA production (either with or without modifications) and purification.
  • a target polynucleotide sequence encoding the polypeptide of interest is first selected for incorporation into a vector, which will be amplified to produce a cDNA template.
  • the target polynucleotide sequence and/or any flanking sequences may be codon optimized.
  • the cDNA template is then used to produce mRNA through in vitro transcription (IVT). After production, the mRNA may undergo purification and clean-up processes. The steps of which are provided in more detail below.
  • the step of gene construction may include, but is not limited to gene synthesis, vector amplification, plasmid purification, plasmid linearization and clean-up, and cDNA template synthesis and clean-up.
  • a primary construct is designed.
  • a first region of linked nucleosides encoding the polypeptide of interest may be constructed using an open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript.
  • the ORF may comprise the wild type ORF, an isoform, variant or a fragment thereof.
  • an "open reading frame” or “ORF” is meant to refer to a nucleic acid sequence (DNA or RNA) that encodes a polypeptide of interest. ORFs often begin with the start codon, ATG, and end with a nonsense or termination codon or signal. Further, the nucleotide sequence of the first region may be codon optimized.
  • Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, adjust translational rates to allow the various domains of the protein to fold properly, or reduce or eliminate problematic secondary structures within the mRNA. Codon optimization tools, algorithms and services are known in the art. Non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In one embodiment, the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are known in the art.
  • the primary constructs of the present disclosure may include at least two stop codons before the 3' untranslated region (UTR).
  • the stop codon may be selected from TGA, TAA and TAG.
  • the primary constructs of the present disclosure include the stop codon TGA and one additional stop codon.
  • the additional stop codon may be TAA.
  • the primary constructs of the present disclosure include three stop codons.
  • the vector containing the primary construct is then amplified and the plasmid isolated and purified using methods known in the art such as, but not limited to, a maxi prep using the Invitrogen PURELINK.TM. HiPure Maxiprep Kit (Carlsbad, Calif.).
  • the plasmid may then be linearized using methods known in the art such as, but not limited to, the use of restriction enzymes and buffers.
  • the linearization reaction may be purified using methods including, for example Invitrogen's PURELINK® PCR Micro Kit (Carlsbad, Calif.), and HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP- HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen's standard PURELINK.TM. PCR Kit (Carlsbad, Calif.). The purification method may be modified depending on the size of the linearization reaction conducted.
  • the linearized plasmid is then used to generate cDNA for in vitro transcription (IVT) reactions.
  • IVT in vitro transcription
  • a cDNA template may be synthesized by having a linearized plasmid undergo polymerase chain reaction (PCR).
  • Primer-probe design for any amplification is within the skill of those in the art.
  • Probes may also contain chemically modified bases to increase base-pairing fidelity to the target molecule and base-pairing strength. Such modifications may include 5-methyl-Cytidine, 2,6-di-amino-purine, 2'-fluoro, phosphoro-thioate, or locked nucleic acids.
  • the process of mRNA or mmRNA production may include, but is not limited to, in vitro transcription, cDNA template removal and RNA clean-up, and mRNA capping and/or tailing reactions.
  • the cDNA produced above may be transcribed using an in vitro transcription (IVT) system.
  • the system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.
  • NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein.
  • the NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.
  • the polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids.
  • the 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability. It binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5' proximal introns during mRNA splicing.
  • Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'-triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule.
  • This 5'-guanylate cap may then be methylated to generate an N7- methyl-guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-0- methylated.
  • 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation. Modifications to the polynucleotides, primary constructs, and mmRNA of the present disclosure may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life.
  • modified nucleotides may be used during the capping reaction.
  • a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with . alpha. -thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.
  • Additional modified guanosine nucleotides may be used such as .alpha.-methyl- phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5'-terminal and/or 5'-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring.
  • Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as an mRNA molecule.
  • UTRs Untranslated regions of a gene are transcribed but not translated.
  • the 5'UTR starts at the transcription start site and continues to the start codon but does not include the start codon, whereas the 3'UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • UTRs can be incorporated into the polynucleotides, primary constructs and/or mRNA of the present disclosure to enhance the stability of the molecule. UTRs also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
  • introns or portions of introns sequences may be incorporated into the flanking regions of the polynucleotides, primary constructs or mmRNA of the disclosure.
  • AU rich elements can be separated into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C- Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) (SEQ ID NO: 38 )nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif.
  • c-Jun and Myogenin are two well-studied examples of this class.
  • Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA.
  • HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
  • Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides, primary constructs or mmRNA of the disclosure.
  • ARE When engineering specific polynucleotides, primary constructs or mmRNA, one or more copies of an ARE can be introduced to make polynucleotides, primary constructs or mmRNA of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using polynucleotides, primary constructs or mmRNA of the disclosure and protein production can be assayed at various time points post-transfection.
  • cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.
  • the polynucleotide, primary construct, and mRNA of the disclosure can be formulated using natural and/or synthetic polymers.
  • Non-limiting examples of polymers which may be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.) formulations from MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM (PHASERX.RTM., Seattle, Wash ), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers.
  • DYNAMIC POLYCONJUGATE® Arrowhead Research Corp., Pasadena, Calif.
  • PHASERXTM polymer formulations such as, without limitation, SMARTT
  • RONDELTM RNAi/Oligonucleotide Nanoparticle Delivery
  • PHASERX® pH responsive co-block polymers
  • Many of these polymer approaches have demonstrated efficacy in delivering oligonucleotides in vivo into the cell cytoplasm (reviewed in deFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated by reference in its entirety).
  • the first of these delivery approaches uses dynamic poly conjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007 104: 12982- 12887; herein incorporated by reference in its entirety).
  • This particular approach is a multicomponent polymer system whose key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and N-acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887; herein incorporated by reference in its entirety).
  • the polymer complex On binding to the hepatocyte and entry into the endosome, the polymer complex disassembles in the low-pH environment, with the polymer exposing its positive charge, leading to endosomal escape and cytoplasmic release of the siRNA from the polymer.
  • the polymer Through replacement of the N-acetylgalactosamine group with a mannose group, it was shown one could alter targeting from asialoglycoprotein receptor-expressing hepatocytes to sinusoidal endothelium and Kupffer cells.
  • Another polymer approach involves using transferrin-targeted cyclodextrin-containing polycation nanoparticles.
  • Mouse otoferlin cDNA transcript variant 4 (KX060996; coding DNA sequence (CDS) identical to reference sequence NM 001313767) that had been subcloned from cochlear cDNA (Strenzke et al. (2016) EMBO J. 35 2519-2535) was subcloned into the backbone for AAV production using standard cloning strategies including restriction digests and ligation. Both vectors contain ITRs of serotype 2. A CMV enhancer and human J-actin promoter were subcloned into the 5’ vector, which contains eGFP cDNA and a P2A signal ( Figure 1).
  • the otoferlin CDS was split at the exon21-exon22 junction into two halves of about similar size.
  • the 5’ vector encodes the N-terminal part of otoferlin from amino acid 1 to 844
  • the 3’ vector contains the coding sequence for amino acids 845 to 1977 and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and poly-adenlyation signals.
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • a splice donor site (Trapani et al. (2014) EMBO Mol. Med. 6(2): 194-211, 2014) follows the coding sequence in the 5’ vector.
  • a splice acceptor site was subcloned just before the coding sequence for otoferlin.
  • a silent mutation was introduced, generating an additional site for restriction digestion.
  • Dual AAV vectors were produced by transient transfection of HEK293 cells grown in multi-level cell factories. The cells were co-transduced with helper plasmids for virus production encoding serotype 6 capsid proteins. Purification of cell lysates was performed by iodixanol density -gradient ultracentrifugation, followed by a second purification and concentration step by FPLC affinity-chromatography (Asai et al. (2015) Nat. Neurosci. 18 1584-1593; Tereshchenko et al. (2014) Neurobiol. Dis. 65 35-42). For the trans-splicing approach, the 5’ vector achieved a concentration of ⁇ 2.8 x 10 8 transducing units/pL. The 3’ vector reached ⁇ 1.4 x 10 8 transducing units/pL. For the hybrid approach, both viruses were purified simultaneously in the same solutions, reaching slightly higher virus titers.
  • Otoferlin knock-out mice were generated as described (Reisinger et al. (2011) J. Neurosci. 31 4886-4895) and backcrossed for at least 5 generations to either C57/B16N or to CD1 strains.
  • FI offspring from Otof ⁇ ⁇ CD1 females and Otof ⁇ ' C57/B16N males were used.
  • Otof +/+ mice from Otof +/ C57/B16N breeding were crossed with CD1 wild type mice (Charles River).
  • mice at postnatal day 6 to 7 were anesthetized using 2.5% - 5% isoflurane.
  • the skin behind the left bulla was opened and the round window niche was exposed.
  • Virus solution in a glass capillary was injected through the round window membrane using a PLI-100A BASIC PICOLITER microinjector (Harvard Apparatus GmbH, Germany) as pressure source, thereby injecting about 0.2-0.5 pL solution per inner ear (Jung et al. (2015) EMBO J. 342686-2702).
  • the skin was closed and the pups were raised by their mothers.
  • Antibodies were diluted in blocking solution and applied to the organ of Corti situated in the temporal bones before apical and basal turns were excised.
  • the following antibodies were used: goat IgGl anti-Ctbp2 mouse anti-otoferlin (RRID:AB_881807, Abeam, Cambridge, UK, 1:300), rabbit anti- otoferlin (Synaptic Systems, Gottingen, Germany, 1:100), goat anti-calbindin D28k and secondary Alexa Fluor®405, Alexa Fluor®488-, Alexa Fluor®568-, Alexa Fluor®594-, and AlexaFluor6474abeled antibodies (Invitrogen, 1:200).
  • the number of synapses in 14-16 day old inner hair cells (IHCs) were counted using the cell counter plugin in ImageJ software as number of Ctbp2 spots.
  • Image analysis to determine fractional levels of membrane bound otoferlin is described in Strenzke et al. (2016a) EMBO J. 35 2519-2535).
  • the pipette solution contained 130 mM Cs-gluconate, 10 mM tetraethylammonium-chloride (TEA-C1), 10 mM 4- aminopyridine (Merck, Darmstadt, Germany), 1 mM MgCb, 10 mM Cs-HEPES (pH 7.17, osmolarity approx. 290 mOsm), 300 pg/mL amphotericin B (Calbiochem, La Jolla, CA).

Abstract

L'invention concerne des compositions comprenant au moins deux vecteurs d'acides nucléiques différents, chacun de ces au moins deux vecteurs différents comprenant une séquence de codage codant pour une partie différente d'une protéine otoferline, ainsi que l'utilisation de ces compositions pour traiter une perte d'audition chez un sujet.
EP21715694.2A 2020-02-21 2021-02-19 Compositions et méthodes de traitement d'une hypoacousie non associée à l'âge chez un sujet humain Pending EP4114958A1 (fr)

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